Multi-directional microfluidic devices comprising a pan-capture binding region and methods of using the same

ABSTRACT

Microfluidic devices and methods for using the same are provided. Aspects of the invention include microfluidic devices that include a separation medium and a pan-capture binding medium. The microfluidic devices are configured to subject a sample to two or more directionally distinct electric fields. Also provided are methods of using the devices as well as systems and kits that include the devices. The devices, systems and methods find use in a variety of different applications, including diagnostic and validation assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S.Provisional Patent Application No. 61/416,693 filed on Nov. 23, 2010,the disclosure of which is herein incorporated by reference in itsentirety.

INTRODUCTION

A variety of analytical techniques may be used to detect specificanalytes in a given sample. For example, Western blotting can be used todetect proteins in a sample by using gel electrophoresis to separate theproteins in the sample followed by probing with antibodies specific forthe target protein. Southern blotting combines transfer ofelectrophoresis-separated DNA fragments to a filter membrane andsubsequent fragment detection by probe hybridization. Northern blottinginvolves the use of electrophoresis to separate RNA samples by size anddetection with a hybridization probe complementary to part of or theentire target sequence. Eastern blotting can be used to detect proteinpost translational modifications (PTM) by analyzingelectrophoresis-separated proteins for post-translational modificationsusing probes specific for lipids, carbohydrate, phosphorylation or anyother protein modifications. Far-Western blotting is similar to Westernblotting, but uses a non-antibody protein to bind the protein ofinterest, and thus can be used to detect protein-protein interactions.Southwestern blotting is a technique that can be used to detectDNA-binding proteins by using gel electrophoresis to separate theproteins in a sample followed by probing with genomic DNA fragments.

Conventional blotting techniques, as discussed above, may fall short ofperformance needs for applications that demand either high-throughputsample analysis or operation in resource poor settings. Blottingtechniques may require labor-intensive, time consuming, multi-stepprocedures carried out by a trained technician, and thus may beimpractical for use in a clinical setting. Furthermore, devices that areless expensive and easier to fabricate and operate are desired.

SUMMARY

Microfluidic devices and methods for using the same are provided.Aspects of the invention include microfluidic devices that include aseparation medium and a pan-capture binding medium. The microfluidicdevices are configured to subject a sample to two or more directionallydistinct flow fields. Also provided are methods of using the devices aswell as systems and kits that include the devices. The devices, systemsand methods find use in a variety of different applications, includingdiagnostic and validation assays.

Aspects of the present disclosure include a microfluidic device fordetecting an analyte in a fluid sample. In certain embodiments, themicrofluidic device includes a separation medium having a separationflow path with a first directional axis, and a pan-capture bindingmedium in fluid communication with the separation medium and having aflow path with a second directional axis. In some cases, the seconddirectional axis is orthogonal to the first directional axis.

The binding medium may be configured to non-specifically bind toanalytes in the sample through electrostatic interactions. For instance,the binding medium may be configured to have a negative charge. In someinstances, the binding medium includes a negatively charged gel. Incertain embodiments, the binding medium includes a negatively chargedpan-capture binding member stably associated with a support. In somecases, the fluid sample includes a detergent. The detergent may beconfigured to provide analytes in the sample with a positive charge.Embodiments that include a negatively charged gel and a detergent thatgives analytes in the sample a positive charge may facilitateelectrostatic binding of the analytes to the binding medium. In certaininstances, the detergent is cetyltrimethylammonium bromide.

In some instances, the analyte includes a fluorescent label. In someinstances, the microfluidic device is configured to subject a sample totwo or more directionally distinct flow fields. For example, the two ormore directionally distinct flow fields may include two or moredirectionally distinct electric fields. In certain cases, themicrofluidic device includes a chamber containing the separation mediumand the binding medium.

Aspects of the present disclosure also include a method of detecting ananalyte in a fluid sample. The method includes: (a) introducing thefluid sample that includes the analyte into a microfluidic deviceconfigured to subject a sample to two or more directionally distinctflow fields; (b) directing the sample through the separation medium toproduce a separated sample; and (c) detecting the analyte in theseparated sample. As indicated above, the microfluidic device includes aseparation medium having a separation flow path with a first directionalaxis, and a pan-capture binding medium in fluid communication with theseparation medium and having a flow path with a second directional axis.

In certain embodiments, the method of detecting an analyte also includestransferring the separated sample to the binding medium. The method mayalso include contacting the analyte with a label that specifically bindsto the analyte to produce a labeled analyte. In some cases, the methodfurther includes detecting the labeled analyte.

In certain instances, the method is a diagnostic method, or in otherinstances may be a validation method.

Aspects of the present disclosure also include a system for detecting ananalyte in a fluid sample. The system includes a microfluidic deviceconfigured to subject a sample to two or more directionally distinctflow fields, and a detector. As described above, the microfluidic deviceincludes a separation medium having a separation flow path with a firstdirectional axis, and a pan-capture binding medium in fluidcommunication with the separation medium and having a flow path with asecond directional axis.

In certain embodiments, the system also includes microfluidic componentsconfigured to direct a fluid through the microfluidic device.

Aspects of the present disclosure also include a kit that includes amicrofluidic device configured to subject a sample to two or moredirectionally distinct flow fields, and a buffer. As described above,the microfluidic device includes a separation medium having a separationflow path with a first directional axis, and a pan-capture bindingmedium in fluid communication with the separation medium and having aflow path with a second directional axis.

In certain embodiments, the buffer includes a detergent configured toprovide analytes in the sample with a positive charge. For example, thedetergent may be cetyltrimethylammonium bromide.

In certain embodiments, the kit also includes one or more reagents, suchas, but not limited to, a detection reagent, a release reagent, adetergent, a refolding reagent and a denaturing reagent.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show a photograph and a schematic drawing of amicrofluidic device, according to embodiments of the present disclosure.

FIG. 2 shows a schematic drawing of a microfluidic device and anenlargement of the chamber that includes a loading medium, a separationmedium and a binding medium, according to embodiments of the presentdisclosure.

FIGS. 3A-3H show schematics of the separation, transfer and detection ofanalytes in a sample, according to embodiments of the presentdisclosure.

FIG. 4 shows a schematic of the addition of a stream of CTAB buffer intothe separation medium by electrophoretic injection, which defines theseparation axis in the microfluidic device, according to embodiments ofthe present disclosure.

FIG. 5 shows a photograph and enlargement of a microfluidic system,according to embodiments of the present disclosure.

FIG. 6 shows schematics of the fabrication steps used forphoto-patterning a polyacrylamide gel inside a microfluidic chamber,according to embodiments of the present disclosure.

FIG. 7 (top) shows a montage of fluorescence images over time (seconds)for the separation of a protein sample in a microfluidic device,according to embodiments of the present disclosure. FIG. 7 (bottom)shows a graph of a linear log-molecular mass (Mr) vs. mobility (10⁻⁵cm²/V·s) relation for the separation of a protein sample in amicrofluidic device, according to embodiments of the present disclosure.

FIGS. 8A-8C show fluorescence images of the separation and transfer offour proteins (protein G, OVA, BSA, and α-actinin) in a sample using amicrofluidic device, according to embodiments of the present disclosure.

FIG. 9A shows a fluorescence image of separated proteins in a sample(protein G, OVA, BSA, and α-actinin) immobilized in the binding mediumof a microfluidic device, according to embodiments of the presentdisclosure. FIG. 9B shows a fluorescence image of subsequent binding ofa detectable label (antibody) to protein G in the separated sample,according to embodiments of the present disclosure.

FIGS. 10A-10D show fluorescence images of separated proteins in a sample(protein G, OVA, BSA, and α-actinin) during separation and binding in amicrofluidic device that included immunoglobulin G (IgG) in the bindingmedium, according to embodiments of the present disclosure.

FIGS. 11A-11D show fluorescence images of separated proteins in a sample(protein G, OVA, BSA, and α-actinin) during separation and binding in amicrofluidic device that included β-galactosidase (β-gal) in the bindingmedium, according to embodiments of the present disclosure.

FIG. 12 shows fluorescence images and corresponding graphs offluorescence intensity for various steps including the separation,transfer, binding (e.g., immobilization), washing and blocking performedduring a method, according to embodiments of the present disclosure.

FIG. 13 shows a graph of protein capture efficiency for various stepsincluding the separation, transfer, binding (e.g., immobilization),washing and blocking performed during a method, according to embodimentsof the present disclosure.

FIG. 14 shows a multispectral composite fluorescence image of separatedsample proteins (protein G, OVA, BSA, and β-gal* (β-galactosidasemonomer)) with protein G labeled with a detectable label (e.g., antibodyprobe), according to embodiments of the present disclosure.

FIG. 15A shows a graph of capture efficiency (%) vs. β-gal concentration(μM) illustrating the effect of charge density of the electrostaticbinding medium, according to embodiments of the present disclosure. FIG.15B shows a graph of capture efficiency (%) vs. ionic strength of thetransfer buffer (mM), according to embodiments of the presentdisclosure.

FIG. 16 shows fluorescence images of the binding strength of proteins indifferent charge densities and binding members (β-gal vs. Immobiline),according to embodiments of the present disclosure.

FIG. 17 shows fluorescence images of the binding strength of proteins indifferent ionic strength buffers (e.g., different bufferconcentrations), according to embodiments of the present disclosure.

FIG. 18 shows schematics of a comparison of charge interaction between aCTAB-protein complex and a) Immobilines, and b) β-gal copolymerized in apolyacrylamide gel pore, according to embodiments of the presentdisclosure.

FIG. 19 shows fluorescence images of CTAB-PAGE separation andimmunoblotting of a sample of proteins and human lactoferrin in: (a)1×TA buffer and (b) human tear fluid, according to embodiments of thepresent disclosure.

DETAILED DESCRIPTION

Microfluidic devices and methods for using the same are provided.Aspects of the invention include microfluidic devices that include aseparation medium and a pan-capture binding medium. The microfluidicdevices are configured to subject a sample to two or more directionallydistinct electric fields. Also provided are methods of using the devicesas well as systems and kits that include the devices. The devices,systems and methods find use in a variety of different applications,including diagnostic and validation assays.

Below, the subject microfluidic devices are described first in greaterdetail. Methods of detecting an analyte in a fluid sample are alsodisclosed in which the subject microfluidic devices find use. Inaddition, systems and kits that include the subject microfluidic devicesare also described.

Microfluidic Devices

Aspects of the present disclosure include microfluidic devices fordetecting an analyte in a fluid sample. A “microfluidic device” isdevice that is configured to control and manipulate fluids geometricallyconstrained to a small scale (e.g., sub-millimeter). Embodiments of themicrofluidic devices include a separation medium and a pan-capturebinding medium. The separation medium may be configured to separateanalytes in a sample from each other. The separated analytes may becontacted with the pan-capture binding medium, which non-specificallybinds to components in the sample. The bound analyte or analytes ofinterest may then be detected. Additional details about the separationmedium and pan-capture binding medium are discussed below.

Separation Medium

In certain embodiments, the microfluidic devices include a separationmedium. The separation medium may be configured to separate analytes ina sample from each other. In some cases, the separation medium isconfigured to separate analytes in a sample based on the physicalproperties of the analytes. For example, the separation medium may beconfigured to separate the analytes in the sample based on the molecularmass, size, charge (e.g., charge to mass ratio), isoelectric point, etc.of the analytes. In certain instances, the separation medium isconfigured to separate the analytes in the sample based on the molecularmass of the analytes. In some cases, the separation medium is configuredto separate the analytes in the sample based on the isoelectric point ofthe analytes (e.g., isoelectric point focusing). The separation mediummay be configured to separate the analytes in the sample into distinctdetectable bands of analytes. By “band” is meant a distinct detectableregion where the concentration of an analyte is significantly higherthan the surrounding regions. Each band of analyte may include a singleanalyte or several analytes, where each analyte in a single band ofanalytes has substantially similar physical properties, as describedabove.

In certain embodiments, the separation medium is configured to separatethe analytes in a sample as the sample traverses the separation medium.In some cases, the separation medium is configured to separate theanalytes in the sample as the sample flows through the separationmedium. Aspects of the separation medium include that the separationmedium has a flow path with a directional axis. By “flow path” is meantthe direction a fluid sample travels as it moves. In some instances, theflow path is the direction the sample travels as the sample traverses amedium, such as the separation medium. As indicated above, theseparation medium may have a flow path with a directional axis. In someembodiments, the directional axis of the separation flow path is alignedwith the length of the separation medium. In these embodiments, thesample traverses the separation medium in the direction of theseparation flow path of the separation medium (e.g., the sample maytraverse the separation medium along the length of the separationmedium). In some cases, the length of the separation medium is greaterthan the width of the separation medium, such as 2 times, 3 times, 4times, 5 times, 10 times, etc. the width of the separation medium. Insome instances, the separation flow path of the separation medium isdefined by a region that includes the separation medium. For example,the microfluidic device may include a chamber. The chamber may include aseparation region that includes the separation medium and a bindingregion that includes the pan-capture binding medium. The separationmedium may be included in the chamber, such that a sample traverses theseparation medium as the sample flows through the chamber.

In certain embodiments, the separation medium includes a polymer, suchas a polymeric gel. The polymeric gel may be a gel suitable for gelelectrophoresis. The polymeric gel may include, but is not limited to, apolyacrylamide gel, an agarose gel, and the like. The resolution of theseparation medium may depend on various factors, such as, but notlimited to, pore size, total polymer content (e.g., total acrylamidecontent), concentration of cross-linker, applied electric field, assaytime, and the like. For instance, the resolution of the separationmedium may depend on the pore size of the separation medium. In somecases, the pore size depends on the total polymer content of theseparation medium and/or the concentration of cross-linker in theseparation medium. In certain instances, the separation medium isconfigured to resolve analytes with molecular mass differences of 50,000Da or less, or 25,000 Da or less, or 10,000 Da or less, such as 7,000 Daor less, including 5,000 Da or less, or 2,000 Da or less, or 1,000 Da orless, for example 500 Da or less, or 100 Da or less. In some cases, theseparation medium may include a polyacrylamide gel that has a totalacrylamide content, T (T=total concentration of acrylamide andbisacrylamide monomer), ranging from 1% to 20%, such as from 3% to 15%,including from 5% to 10%. In some instances, the separation medium has atotal acrylamide content of 6%.

In certain embodiments, the separation medium includes a buffer. Thebuffer may be any convenient buffer used for gel electrophoresis. Incertain embodiments, the buffer is an alkaline buffer, such as, but notlimited to, a tricine-arginine buffer. In certain embodiments, theseparation medium includes a buffer, such as a Tris-glycine buffer. Forexample, the buffer may include a mixture of Tris and glycine.

In some cases, the buffer includes a detergent. In certain instances,the detergent is configured to provide analytes in the sample withsubstantially similar charge-to-mass ratios. Analytes with substantiallysimilar charge-to-mass ratios may facilitate the separation of theanalytes into one or more bands in the separation medium based on themolecular masses of the analytes in the sample. In certain cases, thedetergent is a cationic detergent configured to provide analytes in thesample with a charge, such as a positive charge. For example, thedetergent may be a cationic detergent configured to provide analytes inthe sample with a positive charge. Analytes with a positive charge mayfacilitate electrostatic binding of the analytes to a negatively chargedbinding medium, as discussed in further detail below. In someembodiments, the detergent is cetyltrimethylammonium bromide (CTAB),also known as cetrimonium bromide or hexadecyltrimethylammonium bromide.

In some instances, as described above, the buffer includes a detergent.Certain embodiments of the buffer may include an anionic detergent. Incertain cases, the detergent is an anionic detergent configured toprovide analytes in the sample with a negative charge. Analytes with anegative charge may facilitate electrostatic binding of the analytes toa positively charged binding medium. For instance, the detergent may bean anionic detergent, such as, but not limited to, sodium dodecylsulfate (SDS).

Pan-Capture Binding Medium

Aspects of the microfluidic devices include a pan-capture bindingmedium. By “pan-capture” is meant that the binding mediumnon-specifically binds to analytes in a sample. For example, apan-capture binding medium may non-specifically bind to proteins in asample. Non-specific binding may include binding to substantially all ofthe analytes in a sample. In some cases, non-specific binding is basedon a binding interaction between the analytes in a sample and thepan-capture binding medium. The binding interaction can be based on oneor more of a variety of binding interactions between the pan-capturebinding medium and the analytes in the sample, such as, but not limitedto, covalent bonds, ionic bonds, electrostatic interactions, hydrophobicinteractions, hydrogen bonds, van der Waals forces (e.g., Londondispersion forces), dipole-dipole interactions, combinations thereof,and the like. The binding interactions may be substantially permanent(e.g., requiring a relatively large amount of energy to overcome thebinding interaction, such as with covalent bonds) or may be reversible(e.g., requiring a relatively low amount of energy to disrupt thebinding interaction, such as with dipole-dipole interactions).

In certain embodiments, the pan-capture binding medium is configured tonon-specifically bind to analytes in the sample through electrostaticinteractions. In some cases, electrostatic interactions include bindinginteractions due to the attraction between two oppositely charged ions.For example, electrostatic interactions may be present between apositively charged analyte and a negatively charged binding medium.Similarly, electrostatic interactions may be present between anegatively charged analyte and a positively charged binding medium. Incertain instances, the binding medium is configured to have a negativecharge. As such, the negatively charged binding medium may be configuredto have electrostatic binding interactions with positively chargedanalytes. In other instances, the binding medium is configured to have apositive charge. As such, the negatively charged binding medium may beconfigured to have electrostatic binding interactions with positivelycharged analytes.

In certain cases, the binding medium includes a polymer, such as apolymeric gel or polymeric monolith. By monolith is meant a single,contiguous structure. Monoliths may include a single region with thesame physical and chemical composition, or may include two or moreregions that differ in terms of their physical and chemicalcompositions. The polymeric gel may be a gel suitable for gelelectrophoresis. The polymeric gel may include, but is not limited to, apolyacrylamide gel, an agarose gel, and the like. The polymeric gel mayinclude polymers, such as, but is not limited to, acrylate polymers,alkylacrylate polymers, alkyl alkylacrylate polymers, copolymersthereof, and the like. In some cases, the binding medium may include apolyacrylamide gel that has a total acrylamide content ranging from 1%to 20%, such as from 3% to 15%, including from 5% to 10%. For instance,the binding medium may include a polyacrylamide gel with a totalacrylamide content of 9%.

As described above, in certain embodiments, the binding medium isconfigured to have a negative charge. For instance, the binding mediummay include a negatively charged gel, such as a negatively chargedpolyacrylamide gel. A negatively charged gel may facilitateelectrostatic binding interactions with positively charged analytes. Incertain embodiments, the binding medium includes a buffer. In somecases, the buffer is an alkaline buffer. Alkaline buffers may facilitatethe presence of a negative charge on the binding medium (e.g., thepolyacrylamide gel). In some instances, the buffer is an alkalinebuffer, such as, but not limited to, a tricine-arginine buffer.

In other embodiments, the binding medium is configured to have apositive charge. For instance, the binding medium may include apositively charged gel, such as a positively charged polyacrylamide gel.A positively charged gel may facilitate electrostatic bindinginteractions with negatively charged analytes. As described above, thebinding medium may include a buffer. In some cases, the buffer may beconfigured facilitate the presence of a positive charge on the bindingmedium, which in turn may facilitate electrostatic binding interactionswith negatively charged analytes.

In certain embodiments, the binding medium includes a pan-capturebinding member. The pan-capture binding member may be configured to bindto and retain analytes in a sample. For example, the pan-capture bindingmember may be configured to non-specifically bind to analytes in thesample, such as non-specifically binding to proteins in the sample.Similar to the pan-capture binding medium described above, thepan-capture binding member may be configured to bind to analytes basedon one or more of a variety of binding interactions between thepan-capture binding member and the analytes in the sample, such as, butnot limited to, covalent bonds, ionic bonds, electrostatic interactions,hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g.,London dispersion forces), dipole-dipole interactions, combinationsthereof, and the like. In certain embodiments, the pan-capture bindingmember is configured to non-specifically bind to analytes in the samplethrough electrostatic interactions. For instance, the pan-capturebinding member may be configured to have a negative charge, such thatthe pan-capture binding member non-specifically binds to positivelycharged analytes in the sample. In some instances, the pan-capturebinding member includes a negatively charged compound, such as, but notlimited to, negatively charged acrylamido compounds (e.g., Immobilines).In some cases, the pan-capture binding member includes a peptide orprotein (e.g., a capture protein), such as a negatively charged proteinor peptide. For example, the pan-capture binding member may includeimmunoglobulin-G (IgG), β-galactosidase (β-gal), myosin, derivativesthereof, combinations thereof, and the like. As described above,buffers, such as alkaline buffers (e.g., a tricine-arginine buffer), mayfacilitate the presence of a negative charge on the binding member. Insome instances, an alkaline buffer ionizes acidic amino acid residues toprovide a negative charge on the binding member (e.g., the acidic aminoacid residues may have a lower pK value than the pH of the alkalinebuffer).

In other embodiments, the pan-capture binding member may be configuredto non-specifically bind to analytes in the sample through electrostaticinteractions, such that the pan-capture binding member is configured tohave a positive charge. For example, the pan-capture binding member maybe configured to have a positive charge, such that the pan-capturebinding member non-specifically binds to negatively charged analytes inthe sample.

In certain embodiments, the pan-capture binding member is stablyassociated with a support. By “stably associated” is meant that, understandard conditions, a moiety is bound to or otherwise associated withanother moiety or structure. In certain instances, the support is apolymeric gel, as described above. As such, in certain embodiments, themicrofluidic devices include both a pan-capture binding medium and apan-capture binding member, as described herein. Bonds between thebinding member and the support may include covalent bonds andnon-covalent interactions, such as, but not limited to, ionic bonds,electrostatic interactions, hydrophobic interactions, hydrogen bonds,van der Waals forces (e.g., London dispersion forces), dipole-dipoleinteractions, and the like. In certain embodiments, the binding membermay be covalently bound to the support, such as cross-linked orcopolymerized to the support. For example, the binding member may bebound to the support through a linking group, such as, but not limitedto: a receptor/ligand binding pair; a ligand-binding portion of areceptor; an antibody/antigen binding pair; an antigen-binding fragmentof an antibody; a hapten; a lectin/carbohydrate binding pair; anenzyme/substrate binding pair; a biotin/avidin binding pair; abiotin/streptavidin binding pair; a digoxin/antidigoxin binding pair; aDNA or RNA aptamer binding pair; a peptide aptamer binding pair; and thelike. In some cases, the binding member is bound to the support througha biotin/streptavidin binding pair.

Further Aspects of Embodiments of the Microfluidic Devices

Aspects of the microfluidic devices include embodiments where theseparation medium is in fluid communication with the binding medium. Themicrofluidic device may be configured to direct a sample through theseparation medium first to produce a separated sample. In certainembodiments, the microfluidic device is configured such that theseparation medium and the binding medium are in direct fluidcommunication with each other. For example, the separation medium may bein direct contact with the binding medium. In some cases, the separationmedium and the binding medium are bound to each other, such ascontiguously photopatterned side-by-side. Embodiments where theseparation medium is in direct fluid communication with the bindingmedium may facilitate the transfer of components from the separationmedium to the binding medium with a minimal loss of components. In someinstances, the microfluidic devices are configured such that componentsare quantitatively and reproducibly transferred from the separationmedium to the binding medium.

In certain embodiments, the microfluidic device is configured to directthe separated sample to the binding medium. In other cases, themicrofluidic device is configured such that the separation medium andthe binding medium are in direct fluid communication with each other,such that a sample or analyte can traverse directly from the separationmedium to the binding medium. As described above, the binding medium maybe configured to non-specifically bind to analytes in the sample fordetection of an analyte of interest in the separated sample.

In certain embodiments, the microfluidic devices are multi-directionalmicrofluidic devices. By “multi-directional” is meant more than onedirection, such as two or more directions, three or more directions,four or more directions, etc. In certain cases, two or more directionsare included in a single plane, such that the two or more directions areco-planar. In some instances, the microfluidic devices are configured todirect a fluid in more than one direction (e.g., the microfluidicdevices are multi-directional), such as two or more directions, three ormore directions, four or more directions, etc. For example, themicrofluidic devices may be configured to direct a fluid in twodirections, three directions, four directions, etc. For instance, themicrofluidic devices may be included in a substrate, such that themicrofluidic device is planar. The microfluidic device may be configuredto direct fluids in multiple directions within that plane.

Aspects of the microfluidic devices include a separation medium having aseparation flow path and a pan-capture binding medium in fluidcommunication with the separation medium. The separation medium mayinclude a separation flow path with a first directional axis, whichcorresponds to the direction the sample travels as the sample traversesthe separation medium. The binding medium may have a second flow pathwith a second directional axis. In some instances, the second flow pathis the direction the sample travels as the sample traverses from theseparation medium to the binding medium. The binding medium may have adirectional axis different from the directional axis of the separationmedium. For example, the separation medium may have a first directionalaxis and the binding medium may have a second directional axis, wherethe second directional axis is at an angle of 180 degrees or less withrespect to the first directional axis, such as 150 degrees or less, 135degrees or less, including 120 degrees or less, 90 degrees or less, 60degrees or less, 45 degrees or less, or 30 degrees or less with respectto the first directional axis. In certain embodiments, the seconddirectional axis is orthogonal to the first directional axis, such thatthe separation flow path is at a 90 degree angle with respect to theflow path of the binding medium.

In some instances, the microfluidic device is configured to subject asample to two or more directionally distinct flow fields. By “flowfield” is meant a region where components traverse the region insubstantially the same direction. For example, a flow field may includea region where mobile components move through a medium in substantiallythe same direction. A flow field may include a medium, such as aseparation medium, a binding medium, a loading medium, etc., wherecomponents, such as buffers, analytes, reagents, etc., move through themedium in substantially the same direction. A flow field may be inducedby an applied electric field, a pressure differential, electroosmosis,and the like. In some embodiments, the two or more flow fields may bedirectionally distinct. For example, a first flow field may be alignedwith the directional axis of the separation flow path of the separationmedium. The first flow field may be configured to direct the sample oranalytes through the separation medium along the separation flow path. Asecond flow field may be aligned with the directional axis of the flowpath of the binding medium. In some instances, the second flow field isconfigured to direct the sample or analytes from the separation mediumto the binding medium along the flow path of the binding medium. Thesecond flow field may be configured to direct the sample or analytesfrom the separation medium to the binding medium such that the analytescontact and bind to the binding medium. As described above, in certaininstances, the directional axis of the binding medium flow path isorthogonal to the directional axis of the separation flow path. In theseinstances, the second flow field may be orthogonal to the first flowfield.

In certain embodiments, the microfluidic device is configured to subjecta sample to two or more directionally distinct electric fields. Theelectric fields may facilitate the movement of the sample through themicrofluidic device (e.g., electrokinetic transfer of the sample fromone region of the microfluidic device to another region of themicrofluidic device). The electric fields may also facilitate theseparation of the analytes in the sample by electrophoresis (e.g.,polyacrylamide gel electrophoresis (PAGE)), as described above. Forinstance, the electric field may be configured to direct the analytes ina sample through the separation medium of the microfluidic device. Theelectric field may be configured to facilitate the separation of theanalytes in a sample based on the physical properties of the analytes.For example, the electric field may be configured to facilitate theseparation of the analytes in the sample based on the molecular mass,size, charge (e.g., charge to mass ratio), isoelectric point, etc. ofthe analytes. In certain instances, the electric field is configured tofacilitate the separation of the analytes in the sample based on themolecular mass of the analytes.

In some embodiments, the two or more electric fields may bedirectionally distinct. For example, a first electric field may bealigned with the directional axis of the separation flow path of theseparation medium. The first electric field may be configured to directthe sample or analytes through the separation medium along theseparation flow path. A second electric field may be aligned with thedirectional axis of the flow path of the binding medium. In someinstances, the second electric field is configured to direct the sampleor analytes from the separation medium to the binding medium along theflow path of the binding medium. The second electric field may beconfigured to direct the analytes from the separation medium to thebinding medium such that the analytes contact and bind to the bindingmedium. As described above, in certain instances, the directional axisof the binding medium flow path is orthogonal to the directional axis ofthe separation flow path. In these instances, the second electric fieldmay be orthogonal to the first electric field.

In certain embodiments, the microfluidic device includes one or moreelectric field generators configured to generate an electric field. Theelectric field generator may be configured to apply an electric field tovarious regions of the microfluidic device, such as one or more of theseparation medium, the binding medium, the loading medium, and the like.The electric field generators may be configured to electrokineticallytransport the analytes and components in a sample through the variousmedia in the microfluidic device. In certain instances, the electricfield generators may be proximal to the microfluidic device, such asarranged on the microfluidic device. In some cases, the electric fieldgenerators are positioned a distance from the microfluidic device. Forexample, the electric field generators may be incorporated into a systemfor detecting an analyte, as described in more detail below.

Embodiments of the microfluidic device may be made of any suitablematerial that is compatible with the assay conditions, samples, buffers,reagents, etc. used in the microfluidic device. In some cases, themicrofluidic device is made of a material that is inert (e.g., does notdegrade or react) with respect to the samples, buffers, reagents, etc.used in the subject microfluidic device and methods. For instance, themicrofluidic device may be made of materials, such as, but not limitedto, glass, quartz, polymers, elastomers, paper, combinations thereof,and the like.

In some instances, the microfluidic device includes one or more sampleinput ports. The sample input port may be configured to allow a sampleto be introduced into the microfluidic device. The sample input port maybe in fluid communication with the separation medium. In some instances,the sample input port is in fluid communication with the upstream end ofthe separation medium. The sample input port may further include astructure configured to prevent fluid from exiting the sample inputport. For example, the sample input port may include a cap, valve, seal,etc. that may be, for instance, punctured or opened to allow theintroduction of a sample into the microfluidic device, and thenre-sealed or closed to substantially prevent fluid, including the sampleand/or buffer, from exiting the sample input port.

In certain embodiments, the microfluidic device is substantiallytransparent. By “transparent” is meant that a substance allows visiblelight to pass through the substance. In some embodiments, a transparentmicrofluidic device facilitates detection of analytes bound to thebinding medium, for example analytes that include a detectable label,such as a fluorescent label. In some cases, the microfluidic device issubstantially opaque. By “opaque” is meant that a substance does notallow visible light to pass through the substance. In certain instances,an opaque microfluidic device may facilitate the analysis of analytesthat are sensitive to light, such as analytes that react or degrade inthe presence of light.

In some aspects, the separation medium and the binding medium areprovided in a single common chamber, as illustrated in FIG. 2. In theseembodiments, the microfluidic device includes a chamber. The chamber mayinclude a separation medium and a binding medium. As described above,the separation medium may be in fluid communication, such as in directphysical contact, with the binding medium. In some cases, the separationmedium is bound to the binding medium, such as contiguouslyphotopatterned side-by-side with the binding medium. As such, thechamber may be configured to contain both the separation medium and thebinding medium in fluid communication with each other.

In addition to the separation medium and the binding medium, the chambermay also include a loading medium. The loading medium may be in fluidcommunication with the separation medium. In some instances, the loadingmedium is in direct physical contact with the separation medium. Forexample, the loading medium may be bound to the separation medium, suchas contiguously photopatterned side-by-side with the separation medium.The loading medium may be positioned such that the sample contacts theloading medium before contacting the separation medium. In certainembodiments, the loading medium facilitates contacting a sample with theseparation medium. For instance, the loading medium may be configured toconcentrate the sample before the sample contacts the separation medium.In certain embodiments, the loading medium may include two or moreregions that have different physical and/or chemical properties. Theloading medium may include a loading region and a stacking region. Theloading medium may be configured to include a loading region upstreamfrom a stacking region.

In certain embodiments, the loading medium includes a polymer, such as apolymeric gel. The polymeric gel may be a gel suitable for gelelectrophoresis. The polymeric gel may include, but is not limited to, apolyacrylamide gel, an agarose gel, and the like. In some cases, theloading region includes a polymeric gel with a large pore size. Forexample, the loading region may include a polyacrylamide gel that has atotal acrylamide content of 5% or less, such as 4% or less, including 3%or less, or 2% or less. In some instances, the loading region has atotal acrylamide content of 3%. In some cases, the stacking region ofthe loading medium may be configured to concentrate the sample beforethe sample contacts the separation medium. The stacking region mayinclude a polymeric gel with a smaller pore size than the loadingregion. For example, the stacking region may include a polyacrylamidegel that has a total acrylamide content of ranging from 5% to 10%, suchas from 5% to 9%, including from 5% to 8%, or from 5% to 7%. In someinstances, the stacking region has a total acrylamide content of 6%. Thesmaller pore size of the stacking region may slow the electrophoreticmovement of the sample through the stacking region, thus concentratingthe sample before it contacts the separation medium.

In certain instances, the chamber contains the loading medium, theseparation medium and the binding medium. The chamber may be configuredto contain the loading medium, the separation medium and the bindingmedium such that the loading medium, the separation medium and thebinding medium are in fluid communication with each other, as describedabove. For example, the chamber may include a contiguous polymeric gelmonolith with various regions. Each region of the contiguous polymericgel monolith may have different physical and/or chemical properties. Thecontiguous polymeric gel monolith may include a first region having aloading medium, a second region having a separation medium and a thirdregion having a binding medium. The flow paths of each region of thepolymeric gel monolith may be configured such that a sample firstcontacts the loading medium, then contacts the separation medium, andfinally contacts the binding medium.

In certain embodiments, the polymeric gel monolith has a width rangingfrom 0.1 mm to 5 mm, such as from 0.2 mm to 2.5 mm, including from 0.5mm to 1.5 mm. In some cases, the polymeric gel monolith has a width of 1mm. In some instances, the polymeric gel monolith has a length rangingfrom 0.5 mm to 5 mm, such as from 0.5 mm to 3 mm, including from 1 mm to2 mm. In certain instances, the polymeric gel monolith has a length of1.5 mm. In certain embodiments, the first region of the polymeric gelmonolith that includes the loading medium has a width ranging from 0.1mm to 5 mm, such as from 0.2 mm to 2.5 mm, including from 0.5 mm to 1.5mm. In some cases, the first region of the polymeric gel monolith thatincludes the loading medium has a width of 0.9 mm. In some cases, thefirst region of the polymeric gel monolith that includes the loadingmedium has a length ranging from 0.1 mm to 2 mm, such as from 0.1 mm to1 mm, including from 0.1 mm to 0.5 mm. In certain embodiments, the firstregion of the polymeric gel monolith that includes the loading mediumhas a length of 0.2 mm. In certain instances, the second region of thepolymeric gel monolith that includes the separation medium has a widthranging from 0.1 mm to 5 mm, such as from 0.2 mm to 2.5 mm, includingfrom 0.5 mm to 1.5 mm. In some cases, the second region of the polymericgel monolith that includes the separation medium has a width of 0.9 mm.In some cases, the second region of the polymeric gel monolith thatincludes the separation medium has a length ranging from 0.5 mm to 5 mm,such as from 0.5 mm to 3 mm, including from 1 mm to 2 mm. In certainembodiments, the second region of the polymeric gel monolith thatincludes the separation medium has a length of 1.3 mm. In certaininstances, the third region of the polymeric gel monolith that includesthe binding medium has a width ranging from 0.01 mm to 2 mm, such asfrom 0.01 mm to 1 mm, including from 0.05 mm to 0.5 mm. In some cases,the third region of the polymeric gel monolith that includes the bondingmedium has a width of 0.1 mm. In some cases, the third region of thepolymeric gel monolith that includes the binding medium has a lengthranging from 0.5 mm to 5 mm, such as from 0.5 mm to 3 mm, including from1 mm to 2 mm. In certain embodiments, the third region of the polymericgel monolith that includes the binding medium has a length of 1.5 mm.

In certain embodiments, the microfluidic device has a width ranging from10 cm to 1 mm, such as from 5 cm to 5 mm, including from 1 cm to 5 mm.In some instances, the microfluidic device has a length ranging from 100cm to 1 mm, such as from 50 cm to 1 mm, including from 10 cm to 5 mm, orfrom 1 cm to 5 mm. In certain aspects, the microfluidic device has anarea of 1000 cm² or less, such as 100 cm² or less, including 50 cm² orless, for example, 10 cm² or less, or 5 cm² or less, or 3 cm² or less,or 1 cm² or less, or 0.5 cm² or less, or 0.25 cm² or less, or 0.1 cm² orless.

Further aspects of related microfluidic devices are found in U.S.application Ser. No. 13/055,679, filed Jan. 24, 2011, the disclosure ofwhich is incorporated herein by reference in its entirety.

FIG. 1A shows a photograph of a microfluidic device 10. As shown in FIG.1B, the microfluidic device 10 includes a chamber 11 that contains theloading medium, the separation medium and the binding medium. Themicrofluidic device 10 also includes various microfluidic channels, suchas inlet channels 1, 2, and 3, and control channels 4, 5, 6, 7, and 8.Each microfluidic channel has a corresponding access port (representedby the black dots).

FIG. 2 shows a schematic of a microfluidic device 20 that includes achamber 21 that contains a loading medium 22, a separation medium 23 anda binding medium 24. The loading medium 22 is in fluid communicationwith the separation medium 23, which is in fluid communication with thebinding medium 24. The directional axis of the separation medium 23 isshown by the vertical arrow and indicates the flow path a sampletraverses from the loading medium 22 to the separation medium 23 andalso through the separation medium 23. The directional axis of thebinding medium 24 is shown by the horizontal arrow and indicates theflow path a sample traverses from the separation medium 23 to thebinding medium 24. The microfluidic device 20 also includes variousmicrofluidic channels, such as inlet channels 1, 2, and 3, and controlchannels 4, 5, 6, 7, and 8. Inlet channels 1, 2, and 3 may be configuredto direct a fluid sample into the chamber 21. Control channels 4, 5, 6,7, and 8 may be configured to direct fluids (e.g., reagents, labels,buffers, wash fluids, etc.) into and/or away from the chamber 21. Inaddition, control channels 4, 5, 6, 7, and 8 may be configured to applyan electric field to various regions of the microfluidic device forelectrokinetically transporting analytes in a sample through the loadingmedium 22, the separation medium 23 and the binding medium 24 in thechamber 21.

Methods

Embodiments of the methods are directed to determining whether ananalyte is present in a sample, e.g., determining the presence orabsence of one or more analytes in a sample. In certain embodiments ofthe methods, the presence of one or more analytes in the sample may bedetermined qualitatively or quantitatively. Qualitative determinationincludes determinations in which a simple yes/no result with respect tothe presence of an analyte in the sample is provided to a user.Quantitative determination includes both semi-quantitativedeterminations in which a rough scale result, e.g., low, medium, high,is provided to a user regarding the amount of analyte in the sample andfine scale results in which an exact measurement of the concentration ofthe analyte is provided to the user.

In certain embodiments, the microfluidic devices are configured todetect the presence of one or more analytes in a sample. The methodincludes introducing a fluid sample into a microfluidic device.Introducing the fluid sample into the microfluidic device may includecontacting the sample with the separation medium, or in embodiments ofthe microfluidic devices that include a loading medium, contacting thesample with the loading medium. The method further includes directingthe sample through the separation medium to produce a separated sample.In some cases, the separated sample is produced by gel electrophoresisas the sample traverses the separation medium, as described above. Theseparated sample may include distinct detectable bands of analytes,where each band includes one or more analytes that have substantiallysimilar properties, such as molecular mass, size, charge (e.g., chargeto mass ratio), isoelectric point, etc. depending on the type of gelelectrophoresis performed.

Aspects of the methods may also include transferring the separatedsample to a pan-capture binding medium. In some embodiments, the methodincludes transferring the entire separated sample to the pan-capturebinding medium. In other cases, specific bands of analytes in theseparated sample may be selectively transferred to the binding medium.In some cases, the method includes contacting the analytes in theseparated sample with the pan-capture binding medium. As describedabove, the pan-capture binding medium may be configured tonon-specifically bind to analytes, thus retaining substantially all theanalytes in the binding medium.

In certain embodiments, the method includes detecting an analyte ofinterest bound to the binding medium. Detectable binding of an analyteof interest to the binding medium indicates the presence of the analyteof interest in the sample. In some instances, detecting the analyte ofinterest includes contacting the analyte of interest with a labelconfigured to specifically bind to the analyte of interest. The labelcan be any molecule that specifically binds to a protein or nucleic acidsequence or biomacromolecule that is being targeted (e.g., the analyteof interest). Depending on the nature of the analyte, the label can be,but is not limited to: single strands of DNA complementary to a uniqueregion of the target DNA or RNA sequence for the detection of nucleicacids; antibodies against an epitope of a peptidic analyte for thedetection of proteins and peptides; or any recognition molecule, such asa member of a specific binding pair. For example, suitable specificbinding pairs include, but are not limited to: a member of areceptor/ligand pair; a ligand-binding portion of a receptor; a memberof an antibody/antigen pair; an antigen-binding fragment of an antibody;a hapten; a member of a lectin/carbohydrate pair; a member of anenzyme/substrate pair; biotin/avidin; biotin/streptavidin;digoxin/antidigoxin; a member of a DNA or RNA aptamer binding pair; amember of a peptide aptamer binding pair; and the like. In certainembodiments, the label includes an antibody. The antibody mayspecifically bind to the analyte of interest.

In certain embodiments, the label includes a detectable label.Detectable labels include any convenient label that may be detectedusing the methods and systems, and may include, but are not limited to,fluorescent labels, colorimetric labels, chemiluminescent labels,multicolor reagents, enzyme-linked reagents, avidin-streptavidinassociated detection reagents, radiolabels, gold particles, magneticlabels, and the like. In certain embodiments, the label includes anantibody associated with a detectable label. For example, the label mayinclude a fluorescently labeled antibody that specifically binds to theanalyte of interest.

Samples that may be assayed with the subject methods may include bothsimple and complex samples. Simple samples are samples that include theanalyte of interest, and may or may not include one or more molecularentities that are not of interest, where the number of thesenon-interest molecular entities may be low, e.g., 10 or less, 5 or less,etc. Simple samples may include initial biological or other samples thathave been processed in some manner, e.g., to remove potentiallyinterfering molecular entities from the sample. By “complex sample” ismeant a sample that may or may not have the analyte of interest, butalso includes many different proteins and other molecules that are notof interest. In some instances, the complex sample assayed in thesubject methods is one that includes 10 or more, such as 20 or more,including 100 or more, e.g., 10³ or more, 10⁴ or more (such as 15,000;20,000 or 25,000 or more) distinct (i.e., different) molecular entities,that differ from each other in terms of molecular structure or physicalproperties (e.g., molecular mass, size, charge, isoelectric point,etc.).

In certain embodiments, the samples of interest are biological samples,such as, but not limited to, urine, blood, serum, plasma, saliva, semen,prostatic fluid, nipple aspirate fluid, lachrymal fluid, perspiration,feces, cheek swabs, cerebrospinal fluid, cell lysate samples, amnioticfluid, gastrointestinal fluid, biopsy tissue (e.g., samples obtainedfrom laser capture microdissection (LCM)), and the like. The sample canbe a biological sample or can be extracted from a biological samplederived from humans, animals, plants, fungi, yeast, bacteria, tissuecultures, viral cultures, or combinations thereof using conventionalmethods for the successful extraction of DNA, RNA, proteins andpeptides. In certain embodiments, the sample is a fluid sample, such asa solution of analytes in a fluid. The fluid may be an aqueous fluid,such as, but not limited to water, a buffer, and the like.

As described above, the samples that may be assayed in the subjectmethods may include one or more analytes of interest. Examples ofdetectable analytes include, but are not limited to: nucleic acids,e.g., double or single-stranded DNA, double or single-stranded RNA,DNA-RNA hybrids, DNA aptamers, RNA aptamers, etc.; proteins andpeptides, with or without modifications, e.g., antibodies, diabodies,Fab fragments, DNA or RNA binding proteins, phosphorylated proteins(phosphoproteomics), peptide aptamers, epitopes, and the like; smallmolecules such as inhibitors, activators, ligands, etc.; oligo orpolysaccharides; mixtures thereof; and the like.

In certain embodiments, the method includes concentrating, diluting, orbuffer exchanging the sample prior to directing the sample through theseparation medium. Concentrating the sample may include contacting thesample with a concentration medium prior to contacting the sample withthe separation medium. The concentration medium may include a small poresize polymeric gel, a membrane (e.g., a size exclusion membrane),combinations thereof, and the like. Concentrating the sample prior tocontacting the sample with the separation medium may facilitate anincrease in the resolution between the bands of analytes in theseparated sample because each separated band of analyte may disperseless as the sample traverses through the separation medium. Diluting thesample may include contacting the sample with additional buffer prior tocontacting the sample with the separation medium. Buffer exchanging thesample may include contacting the sample with a buffer exchange mediumprior to contacting the sample with the separation medium. The bufferexchange medium may include a buffer different from the sample buffer.The buffer exchange medium may include, but is not limited to, amolecular sieve, a porous resin, and the like.

In certain embodiments, the method includes contacting the separatedanalytes bound to the binding medium with a blocking reagent prior todetecting the analyte of interest. In some cases, contacting theseparated analytes with a blocking reagent prior to detecting theanalyte of interest may facilitate a minimization in non-specificbinding of a detectable label to the separated analytes. For example,contacting the separated analytes with the blocking reagent prior todetecting the analyte of interest may facilitate a minimization innon-specific binding of a labeled antibody to the separated analytes.The blocking reagent can be any blocking reagent that functions asdescribed above, and may include, but is not limited to, bovine serumalbumin (BSA), non-fat dry milk, casein, and gelatin. In certainembodiments, the method also includes optional washing steps, which maybe performed at various times before, during and after the other stepsin the method. For example, a washing step may be performed aftertransferring the separated sample from the separation medium to thebinding medium, after contacting the separated sample with the blockingreagent, after contacting the separated sample with the detectablelabel, etc.

Embodiments of the method may also include releasing the analyte boundto the binding medium. The releasing may include contacting the boundanalyte with a releasing agent. The releasing agent may be configured todisrupt the binding interaction between the analyte and the bindingmedium. In some cases, the releasing agent is a reagent, buffer, or thelike, that disrupts the binding interaction between the analyte and thebinding medium causing the binding medium to release the analyte. Afterreleasing the analyte from the binding medium, the method may includetransferring the analyte away from the binding medium. For example, themethod may include directing the released analyte downstream from thebinding medium for secondary analysis with a secondary analysis devicesuch as, but is not limited to, a UV spectrometer, and IR spectrometer,a mass spectrometer, an HPLC, an affinity assay device, and the like.

In some embodiments, the methods include the uniplex analysis of ananalyte in a sample. By “uniplex analysis” is meant that a sample isanalyzed to detect the presence of one analyte in the sample. Forexample, a sample may include a mixture of an analyte of interest andother molecular entities that are not of interest. In some cases, themethods include the uniplex analysis of the sample to determine thepresence of the analyte of interest in the sample mixture.

Certain embodiments include the multiplex analysis of two or moreanalytes in a sample. By “multiplex analysis” is meant that the presencetwo or more distinct analytes, in which the two or more analytes aredifferent from each other, is determined. For example, analytes mayinclude detectable differences in their molecular mass, size, charge(e.g., mass to charge ratio), isoelectric point, and the like. In someinstances, the number of analytes is greater than 2, such as 4 or more,6 or more, 8 or more, etc., up to 20 or more, e.g., 50 or more,including 100 or more, distinct analytes. In certain embodiments, themethods include the multiplex analysis of 2 to 100 distinct analytes,such as 4 to 50 distinct analytes, including 4 to 20 distinct analytes.In certain embodiments, multiplex analysis also includes the use of twoor more different detectable labels. The two or more differentdetectable labels may specifically bind to the same or differentanalytes. In some cases, the two or more different detectable labels mayspecifically bind to the same analyte. For instance, the two or moredifferent detectable labels may include different antibodies specificfor different epitopes on the same analyte. The use of two or moredetectable labels specific for the same analyte may facilitate thedetection of the analyte by improving the signal-to-noise ratio. Inother cases, the two or more different detectable labels mayspecifically bind to different analytes. For example, the two or moredetectable labels may include different antibodies specific for epitopeson different analytes. The use of two or more detectable labels eachspecific for different analytes may facilitate the detection of two ormore respective analytes in the sample in a single assay.

In certain embodiments, the method is an automated method. As such, themethod may include a minimum of user interaction with the microfluidicdevices and systems after introducing the sample into the microfluidicdevice. For example, the steps of directing the sample through theseparation medium to produce a separated sample and transferring theseparated sample to the binding medium may be performed by themicrofluidic device and system, such that the user need not manuallyperform these steps. In some cases, the automated method may facilitatea reduction in the total assay time. For example, embodiments of themethod, including the separation and detection of analytes in a sample,may be performed in 30 min or less, such as 20 min or less, including 15min or less, or 10 min or less, or 5 min or less, or 2 min or less, or 1min or less.

FIGS. 3A-3G show schematics of an embodiment of a method for detectingthe presence of an analyte in a sample. The method includespolyacrylamide gel electrophoresis (PAGE) followed by post-separationsample transfer and, finally, detection using a labeled antibody probe.Analytes are electrokinetically transferred from a PAGE separationmedium to a contiguous pan-capture binding medium and are identified insitu by specific affinity interactions. In step 1 (FIG. 3A), a sample 30is contacted with the loading medium 31. The sample includescetyltrimethylammonium bromide (CTAB), which binds to analytes (e.g.,proteins) in the sample at an equal molar ratio to produce analytes withsubstantially the same charge-to-mass ratio. As such, a linearlog-molecular mass (Mr) to mobility relation is observed for theseparation of CTAB-treated proteins (see FIG. 7 (bottom)). In addition,CTAB-treated proteins have a positive charge, which may facilitateelectrostatic binding of the proteins in the sample to the negativelycharged pan-capture binding medium. After the sample 30 is contactedwith the loading medium 31, an electric field is applied along thedirectional axis of the separation medium to direct the sample throughthe loading medium 31 to the interface between the loading medium 31 andthe separation medium 32 (FIGS. 3A-3B). In step 2 (FIGS. 3B-3C), thevarious analytes in the sample are separated by electrophoresis throughthe separation medium 32. The separation medium 32 has a separation flowpath with a first directional axis. An electric field is applied alongthe first directional axis (indicated by the vertical arrow) to directthe sample through the separation medium 32 (FIG. 3B). In step 3 (FIGS.3C-3D), the separated analytes 33 can be transferred to the pan-capturebinding medium 34 by applying an electric field along a seconddirectional axis (indicated by the horizontal arrow) to direct theseparated analytes 33 to the pan-capture binding medium 34 (FIG. 3C).The pan-capture binding medium includes an alkaline buffer (e.g.,tricine-arginine buffer) and is negatively charged. The positivelycharged separated analytes electrostatically bind to the negativelycharged binding medium (FIG. 3D). In step 4 (FIG. 3E), a blockingreagent (e.g., BSA) is contacted with the separated analytes bound tothe binding medium. In some cases, the blocking reagent facilitates aminimization in non-specific binding of a labeled antibody to theseparated analytes. After contacting the blocking reagent with theseparated analytes, the blocking reagent may be washed away (FIG. 3F).In step 5 (FIG. 3G), a detectable label (e.g., a fluorescently labeledantibody) is contacted with the separated analytes bound to the bindingmedium. The detectable label specifically binds to the analyte ofinterest 35 (e.g., the target protein). Unbound label is washed away tofacilitate a reduction in background signal (FIG. 3H). A positivedetection of the detectable label indicates the presence of the analyteof interest 35 in the sample.

Systems

Aspects of certain embodiments include a system for detecting an analytein a sample. In some instances, the system includes a microfluidicdevice as described herein. The system may also include a detector. Insome cases, the detector is a detector configured to detect a detectablelabel. The detector may include any type of detector configured todetect the detectable label used in the assay. As described above,detectable label may be a fluorescent label, colorimetric label,chemiluminescent label, multicolor reagent, enzyme-linked reagent,avidin-streptavidin associated detection reagent, radiolabel, goldparticle, magnetic label, etc. In some instances, the detectable labelis a fluorescent label. In these instances, the detector may beconfigured to contact the fluorescent label with electromagneticradiation (e.g., visible, UV, x-ray, etc.), which excites thefluorescent label and causes the fluorescent label to emit detectableelectromagnetic radiation (e.g., visible light, etc.). The emittedelectromagnetic radiation may be detected by the detector to determinethe presence of the analyte bound to the binding medium.

In some instances, the detector may be configured to detect emissionsfrom a fluorescent label, as described above. In certain cases, thedetector includes a photomultiplier tube (PMT), a charge-coupled device(CCD), an intensified charge-coupled device (ICCD), a complementarymetal-oxide-semiconductor (CMOS) sensor, a visual colorimetric readout,a photodiode, and the like.

Systems of the present disclosure may include various other componentsas desired. For example, the systems may include fluid handlingcomponents, such as microfluidic fluid handling components. The fluidhandling components may be configured to direct one or more fluidsthrough the microfluidic device. In some instances, the fluid handlingcomponents are configured to direct fluids, such as, but not limited to,sample solutions, buffers (e.g., electrophoresis buffers, wash buffers,release buffers, etc.), and the like. In certain embodiments, themicrofluidic fluid handling components are configured to deliver a fluidto the separation medium of the microfluidic device, such that the fluidcontacts the separation medium. The fluid handling components mayinclude microfluidic pumps. In some cases, the microfluidic pumps areconfigured for pressure-driven microfluidic handling and routing offluids through the microfluidic devices and systems disclosed herein. Incertain instances, the microfluidic fluid handling components areconfigured to deliver small volumes of fluid, such as 1 mL or less, suchas 500 μL or less, including 100 μL or less, for example 50 μL or less,or 25 μL or less, or 10 μL or less, or 5 μL or less, or 1 μL or less.

In certain embodiments, the systems include one or more electric fieldgenerators. An electric field generator may be configured to apply anelectric field to various regions of the microfluidic device. The systemmay be configured to apply an electric field such that the sample iselectrokinetically transported through the microfluidic device. Forexample, the electric field generator may be configured to apply anelectric field to the separation medium. In some cases, the appliedelectric field may be aligned with the directional axis of theseparation flow path of the separation medium. As such, the appliedelectric field may be configured to electrokinetically transport theanalytes and components in a sample through the separation medium. Incertain embodiments, the system includes an electric field generatorconfigured to apply an electric field such that analytes and/orcomponents in the sample are electrokinetically transported from theseparation medium to the binding medium. For instance, an appliedelectric field may be aligned with the directional axis of the flow pathof the binding medium. In some cases, the applied electric field isconfigured to electrokinetically transport selected analytes that havebeen separated by the separation medium. Analytes that have beenseparated by the separation medium may be transported to the bindingmedium by applying an appropriate electric field along the directionalaxis of the flow path of the binding medium. In some instances, theelectric field generators are configured to apply an electric field witha strength ranging from 10 V/cm to 1000 V/cm, such as from 100 V/cm to800 V/cm, including from 200 V/cm to 600 V/cm.

In certain embodiments, the electric field generators include voltageshaping components. In some cases, the voltage shaping components areconfigured to control the strength of the applied electric field, suchthat the applied electric field strength is substantially uniform acrossthe separation medium and/or the binding medium. The voltage shapingcomponents may facilitate an increase in the resolution of the analytesin the sample. For instance, the voltage shaping components mayfacilitate a reduction in non-uniform movement of the sample through theseparation medium. In addition, the voltage shaping components mayfacilitate a minimization in the dispersion of the bands of analytes asthe analytes traverses the separation medium.

In certain embodiments, the subject system is a biochip (e.g., abiosensor chip). By “biochip” or “biosensor chip” is meant amicrofluidic system that includes a substrate surface which displays twoor more distinct microfluidic devices on the substrate surface. Incertain embodiments, the microfluidic system includes a substratesurface with an array of microfluidic devices.

An “array” includes any two-dimensional or substantially two-dimensional(as well as a three-dimensional) arrangement of addressable regions,e.g., spatially addressable regions. An array is “addressable” when ithas multiple devices positioned at particular predetermined locations(e.g., “addresses”) on the array. Array features (e.g., devices) may beseparated by intervening spaces. Any given substrate may carry one, two,four or more arrays disposed on a front surface of the substrate.Depending upon the use, any or all of the arrays may be the same ordifferent from one another and each may contain multiple distinctmicrofluidic devices. An array may contain one or more, including two ormore, four or more, eight or more, 10 or more, 25 or more, 50 or more,or 100 or more microfluidic devices. In certain embodiments, themicrofluidic devices can be arranged into an array with an area of 100cm² or less, 50 cm² or less, or 25 cm² or less, 10 cm² or less, 5 cm² orless, such as 1 cm² or less, including 50 mm² or less, 20 mm² or less,such as 10 mm² or less, or even smaller. For example, microfluidicdevices may have dimensions in the range of 10 mm×10 mm to 200 mm×200mm, including dimensions of 100 mm×100 mm or less, such as 50 mm×50 mmor less, for instance 25 mm×25 mm or less, or 10 mm×10 mm or less, or 5mm×5 mm or less, for instance, 1 mm×1 mm or less.

Arrays of microfluidic devices may be arranged for the multiplexanalysis of samples. For example, multiple microfluidic devices may bearranged in series, such that a sample may be analyzed for the presenceof several different analytes in a series of microfluidic devices. Incertain embodiments, multiple microfluidic devices may be arranged inparallel, such that two or more samples may be analyzed at substantiallythe same time.

Aspects of the systems include that the microfluidic devices may beconfigured to consume a minimum amount of sample while still producingdetectable results. For example, the system may be configured to use asample volume of 100 μL or less, such as 75 μL or less, including 50 μLor less, or 25 μL or less, or 10 μL or less, for example, 5 μL or less,2 μL or less, or 1 μL or less while still producing detectable results.In certain embodiments, the system is configured to have a detectionsensitivity of 1 nM or less, such as 500 pM or less, including 100 pM orless, for instance, 1 pM or less, or 500 fM or less, or 250 fM or less,such as 100 fM or less, including 50 fM or less, or 25 fM or less, or 10fM or less. In some instances, the system is configured to be able todetect analytes at a concentration of 1 μg/mL or less, such as 500 ng/mLor less, including 100 ng/mL or less, for example, 10 ng/mL or less, or5 ng/mL or less, such as 1 ng/mL or less, or 0.1 ng/mL or less, or 0.01ng/mL or less, including 1 pg/mL or less. In certain embodiments, thesystem has a dynamic range from 10⁻¹⁸ M to 10 M, such as from 10⁻¹⁵ M to10⁻³ M, including from 10⁻¹² M to 10⁻⁶ M.

In certain embodiments, the microfluidic devices are operated at atemperature ranging from 1° C. to 100° C., such as from 5° C. to 75° C.,including from 10° C. to 50° C., or from 20° C. to 40° C. In someinstances, the microfluidic devices are operated at a temperatureranging from 35° C. to 40° C.

FIG. 5 shows a photograph and enlargement of a microfluidic system. Incertain embodiments, the microfluidic system includes a microfluidicdevice 500 positioned in a fluorescence microscope 501. The fluorescencemicroscope 501 is operatively connected to a light source 503 and adetector 502. In some instances, the system includes a computer 505configured to control the various components of the system whenperforming an assay. The computer 505 may also be configured to storeand analyze data produced by the assay. In certain cases, the systemincludes an electric field generator 504, such as a high-voltagesequencer. The electric field generator 504 may be operatively connectedto the microfluidic device 500 by leads 507 that include electrodes 508.The microfluidic device 500 may also a guide wire 509 for positioningthe microfluidic device 500 in the fluorescence microscope 501. Inaddition, the microfluidic device 500 may be fluidically connected to amanifold 510 configured to direct a flow of a fluid (e.g., sample fluid,buffer, detectable label, reagent, etc.) to the microfluidic device 500.Samples, buffers, detectable labels, reagents, etc. may be contained intest tubes 506 for use in the assays.

Utility

The subject devices, systems and methods find use in a variety ofdifferent applications where determination of the presence or absence,and/or quantification of one or more analytes in a sample is desired.For example, the subject devices, systems and methods find use in theseparation and detection of proteins, peptides, nucleic acids, and thelike. In some cases, the subject devices, systems and methods find usein the separation and detection of proteins. In certain instances, theproteins are native proteins (e.g., non-denatured proteins). Forinstance, the microfluidic devices may include a separation mediumconfigured to separate native proteins without denaturing the proteins.In some cases, the separation medium is configured to separate proteinsunder non-denaturing conditions, such as by using reagents, buffers,detergents, etc., that do not cause significant denaturing of theproteins in the sample. For example, the separation medium may include anon-denaturing detergent, such as, but not limited to,cetyltrimethylammonium bromide (CTAB). The use of non-denaturingconditions may simplify the overall separation and detection process byeliminating the need for renaturing the proteins in the sample.

In certain embodiments, the subject devices, systems and methods finduse in the detection of nucleic acids, proteins, or other biomoleculesin a sample. The methods may include the detection of a set ofbiomarkers, e.g., two or more distinct protein biomarkers, in a sample.For example, the methods may be used in the rapid, clinical detection oftwo or more disease biomarkers in a biological sample, e.g., as may beemployed in the diagnosis of a disease condition in a subject, or in theongoing management or treatment of a disease condition in a subject,etc. In addition, the subject devices, systems and methods may find usein protocols for the detection of an analyte in a sample, such as, butnot limited to, Western blotting, Southern blotting, Northern blotting,Eastern, Far-Western blotting, Southwestern blotting, and the like.

In certain embodiments, the subject devices, systems and methods finduse in detecting biomarkers. In some cases, the subject devices, systemsand methods may be used to detect the presence or absence of particularbiomarkers, as well as an increase or decrease in the concentration ofparticular biomarkers in blood, plasma, serum, or other bodily fluids orexcretions, such as but not limited to urine, blood, serum, plasma,saliva, semen, prostatic fluid, nipple aspirate fluid, lachrymal fluid,perspiration, feces, cheek swabs, cerebrospinal fluid, cell lysatesamples, amniotic fluid, gastrointestinal fluid, biopsy tissue, and thelike.

The presence or absence of a biomarker or significant changes in theconcentration of a biomarker can be used to diagnose disease risk,presence of disease in an individual, or to tailor treatments for thedisease in an individual. For example, the presence of a particularbiomarker or panel of biomarkers may influence the choices of drugtreatment or administration regimes given to an individual. Inevaluating potential drug therapies, a biomarker may be used as asurrogate for a natural endpoint such as survival or irreversiblemorbidity. If a treatment alters the biomarker, which has a directconnection to improved health, the biomarker can serve as a surrogateendpoint for evaluating the clinical benefit of a particular treatmentor administration regime. Thus, personalized diagnosis and treatmentbased on the particular biomarkers or panel of biomarkers detected in anindividual are facilitated by the subject devices, systems and methods.Furthermore, the early detection of biomarkers associated with diseasesis facilitated by the high sensitivity of the subject devices andsystems, as described above. Due to the capability of detecting multiplebiomarkers on a single chip, combined with sensitivity, scalability, andease of use, the presently disclosed microfluidic devices, systems andmethods find use in portable and point-of-care or near-patient moleculardiagnostics.

In certain embodiments, the subject devices, systems and methods finduse in detecting biomarkers for a disease or disease state. In certaininstances, the subject devices, systems and methods find use indetecting biomarkers for the characterization of cell signaling pathwaysand intracellular communication for drug discovery and vaccinedevelopment. For example, the subject devices, systems and methods maybe used to detect and/or quantify the amount of biomarkers in diseased,healthy or benign samples. In certain embodiments, the subject devices,systems and methods find use in detecting biomarkers for an infectiousdisease or disease state. In some cases, the biomarkers can be molecularbiomarkers, such as but not limited to proteins, nucleic acids,carbohydrates, small molecules, and the like.

The subject devices, systems and methods find use in diagnostic assays,such as, but not limited to, the following: detecting and/or quantifyingbiomarkers, as described above; screening assays, where samples aretested at regular intervals for asymptomatic subjects; prognosticassays, where the presence and or quantity of a biomarker is used topredict a likely disease course; stratification assays, where asubject's response to different drug treatments can be predicted;efficacy assays, where the efficacy of a drug treatment is monitored;and the like.

The subject devices, systems and methods also find use in validationassays. For example, validation assays may be used to validate orconfirm that a potential disease biomarker is a reliable indicator ofthe presence or absence of a disease across a variety of individuals.The short assay times for the subject devices, systems and methods mayfacilitate an increase in the throughput for screening a plurality ofsamples in a minimum amount of time.

In some instances, the subject devices, systems and methods can be usedwithout requiring a laboratory setting for implementation. In comparisonto the equivalent analytic research laboratory equipment, the subjectdevices and systems provide comparable analytic sensitivity in aportable, hand-held system. In some cases, the mass and operating costare less than the typical stationary laboratory equipment. The subjectsystems and devices may be integrated into a single apparatus, such thatall the steps of the assay, including separation, transfer, labeling anddetecting of an analyte of interest, may be performed by a singleapparatus. For example, in some instances, there are no separateapparatuses for separation, transfer, labeling and detecting of ananalyte of interest. In addition, the subject systems and devices can beutilized in a home setting for over-the-counter home testing by a personwithout medical training to detect one or more analytes in samples. Thesubject systems and devices may also be utilized in a clinical setting,e.g., at the bedside, for rapid diagnosis or in a setting wherestationary research laboratory equipment is not provided due to cost orother reasons.

Kits

Aspects of the present disclosure additionally include kits that have amicrofluidic device as described in detail herein. The kits may furtherinclude a buffer. For instance, the kit may include a buffer, such as anelectrophoresis buffer, a sample buffer, and the like. In certain cases,the buffer is an alkaline buffer, such as, but not limited to, atricine-arginine buffer. In some instances, the buffer includes adetergent (such as SDS or CTAB), which is employed in the pan-capture ofseparated proteins, as described herein. For example, the buffer mayinclude cetyltrimethylammonium bromide (CTAB).

The kits may further include additional reagents, such as but notlimited to, release reagents, denaturing reagents, refolding reagents,detergents, detectable labels (e.g., fluorescent labels, colorimetriclabels, chemiluminescent labels, multicolor reagents, enzyme-linkedreagents, detection reagents (e.g., avidin-streptavidin associateddetection reagents), calibration standards, radiolabels, gold particles,magnetic labels, etc.), and the like.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Another means would be a computer readable medium, e.g.,diskette, CD, DVD, Blu-Ray, computer-readable memory, etc., on which theinformation has been recorded or stored. Yet another means that may bepresent is a website address which may be used via the Internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

As can be appreciated from the disclosure provided above, embodiments ofthe present invention have a wide variety of applications. Accordingly,the examples presented herein are offered for illustration purposes andare not intended to be construed as a limitation on the invention in anyway. Those of ordinary skill in the art will readily recognize a varietyof noncritical parameters that could be changed or modified to yieldessentially similar results. Thus, the following examples are put forthso as to provide those of ordinary skill in the art with a completedisclosure and description of how to make and use the present invention,and are not intended to limit the scope of what the inventors regard astheir invention nor are they intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by mass,molecular mass is mass average molecular mass, temperature is in degreesCelsius, and pressure is at or near atmospheric.

EXAMPLES Example 1 Design of Microfluidic Device

A glass microfluidic chip (10 mm×15 mm) (FIG. 1A) was designed usingAutocad software (Autodesk, San Rafael; CA). A rectangular microfluidicchamber (1 mm×1.5 mm×0.02 mm) was designed to accommodate threefunctional gel regions for different Western blotting steps. FIG. 2shows a schematic of the microfluidic chamber that included the loadinggel for sample loading, the separation gel for protein sizing, and theblotting gel for transfer and target probing. The chamber was connectedwith several microfluidic channels, such as the injection channels andcontrol channels. The width of each injection channel was 25 μm, and thewidth of each control channel was 10 μm. The injection channel wasbranched into three channels (see channels 1, 2, and 3 in FIG. 2) thatformed a double-T junction, where a narrow sample plug was formed byapplying a pinch current. Control channels (see channels 4, 5, 6, 7, and8 in FIG. 2) were also linked to the chamber. Constant currents wereapplied via these resistive narrow channels from a current source. Theelectric fields were parallel to the currents. The fields werecontrolled to be parallel to the field from the injection channel thatwas used to transfer the sample plug into the chamber. Controlling thefields facilitated a minimization in sample dispersion in the chamber.Applying current in this manner facilitated transfer of the proteinshorizontally and vertically while preserving the separation pattern.

Fabrication

To prepare the microfluidic chips, the glass microfluidic chips werelithographed, isotropically etched, and diced (Caliper Life Sciences,Hopkinton, Mass.). The etch depth was 20 μm. Eight access holes weredrilled using glass drill bits. The chip was cleaned in a piranha bathand thermally bonded to a blank chip for 6 hours at 592° C. in aprogrammable oven.

Before photo-patterning of polyacrylamide (PA) gels inside themicrofluidic chamber, the glass chip was cleaned by flushing themicrofluidic channels with 0.1 M NaOH for 10 min, and rinsing withdeionized (DI) water for 10 min and methanol for 5 min. For covalentlinkage of the PA gel to the inner glass surface, a salination solution,2:2:3:3 ratio of 3-(trimethoxysilyl) propyl methacrylate, glacial aceticacid, DI water, and methanol (all from Sigma, St. Luis, Mo.) wasintroduced into the chip and incubated for 30 min, and rinsed withmethanol for 30 min. For photo-patterning of three functional gels, eachgel precursor solution was introduced inside the chamber by capillaryaction, polymerized using UV light, and then evacuated and exchangedwith subsequent precursor solution using vacuum suction. FIG. 6 shows aschematic drawing of the fabrication steps used for photo-patterning apolyacrylamide gel inside a microfluidic chamber. The 9% T (T=totalacrylamide content) precursor solution for the binding medium gel wasprepared using 30 μL of 30% T acrylamide/bisacrylamide (Sigma), 10 μL of1 mg/mL streptavidin acrylamide (Invitrogen, Carlsbad, Calif.), 40 μL of2 mg/mL of the binding member (e.g., capture protein), 5×TA buffer(1×TA: 25 mM tricine-14 mM arginine, all from Sigma), and 5 μL of 1%(v/w) photoinitiator VA-086 solution (Wako Chemical, Richmond, Va.). The6% T separation gel and 3% T loading gel were photopatterned similarlyexcept that streptavidin-acrylamide and the binding member (e.g.,capture protein) were not included in polyacrylamide gel precursors. 3%T gel was also polymerized in the microfluidic injection channels forlater sample loading.

A UV exposure system was used for photo-patterning. An invertedmicroscope (IX-50, Olympus, Melville, N.Y.) equipped with mercury-lampand UV objective lens (UPLANS-APO 4×, Olympus) allowed for manualalignment between a glass chip and a transparency mask(Fineline-Imaging, Colorado Springs, Colo.) and UV exposure. The bindingand separation gels were exposed for 8 min at 13 mW/cm². The loading gelwas blanket-exposed for 6 min at 9 mW/cm² under a UV lamp (Blak-Ray,Upland, Calif.).

Four molecular mass standards were conjugated with fluorescein (AlexaFluor 488, Invitrogen): 800 nM protein G (20 kDa, Abcam, Cambridge,Mass.), 200 nM ovalbumin (OVA, 45 kDa, Sigma), 200 nM bovine serumalbumin (BSA, 68 kDa, Sigma), and 500 nM α-actinin (95 kDa, Sigma). Theproteins were solubilized in 1×TA buffer. 0.1% CTAB (Sigma) was added tothe proteins 5 min before loading to the chip.

Microfluidic PAGE Assay

All assay steps of microchip Western blotting (see FIGS. 3A-3G) wereperformed by controlling voltage and current via the eight access ports(see FIG. 2) with a high-voltage sequencer (Caliper Life Sciences). Thevoltage/current sequence is shown in Table 1.

TABLE 1 Voltage/Current Program for Western Blotting Assay Steps Assaysteps/ access port # 1 2 3 4 5 6 7 8 Plug formation −261 V −350 V −200 V   0 A    0 A    0 A    0 A −267 V Sample loading/ −200 V −320 V −320 V−270 V −270 V    0 A    0 A −400 V Separation Electrotransfer    0 A   0 A    0 A    0 A    0 A −200 V −100 V    0 A Blocking/Immuno-    0 A   0 A    0 A    0 A    0 A −200 V −300 V    0 A detection

Prior to the sample loading step, CTAB was electrophoreticallyintroduced in PA gels (see FIG. 4). Pre-treating the PA gels with CTABmay facilitate separation of the analytes in the sample because withoutsufficient CTAB concentration in the gels: (1) protein may lose CTABions that were previously bound in the sample preparation step due todilution, and (2) electrostatic interaction between negatively chargedPA gel and positively charged CTAB-treated protein may result in bindingof proteins before the PAGE separation. Therefore, 0.1% CTAB wasinjected into microchannels from the access ports 1, 2 and 3 for 10 min,and injected into the microfluidic chamber for 5 min as a narrow bandthat was wide enough for a protein plug to migrate without non-specificinteraction.

An epi fluorescence microscope (IX-7, Olympus) equipped withPeltier-cooled CCD camera (CoolSNAP HQ2, Photometrics, Tucson, Ariz.)was used to capture fluorescence images of proteins during all assaystages. The images were later analyzed for protein quantitation usingImageJ software (NIH, Bethesda, Md.).

Results

The CTAB-treated molecular-mass standard was pipetted into the accesshole 3. A narrow plug was formed at the double-T junction. Due to largepore size (3% T) sample loading in the loading gel required 1 min orless. The sample was injected into the microfluidic chamber (FIG. 3A).Upon reaching the separation gel (6% T), the decrease in pore sizeresulted in sample stacking at the loading gel-separation gel interface(FIG. 3B). While migrating downstream, the protein mixture separatedinto multiple bands based on the molecular mass of the proteins due tosieving action in the PA gel (FIG. 3C). As seen in FIG. 7, top, aprotein sample that included α-actinin, BSA, OVA and protein G stackedand then separated into compact bands within 30 s. A linearlog-molecular mass (log-Mr) vs. mobility (10⁻⁵ cm²/V·s) relation wasobserved (see FIG. 7, bottom), indicating that Mr determination usingCTAB-PAGE was accurate.

After separation, a horizontal electric field was applied to transferthe separated protein bands to the binding gel (FIGS. 3C-3D). Uponreaching the binding gel, the separated protein bands were compressedand immobilized due to electrostatic interaction with PA gel (FIG. 3D).FIGS. 7A, 7B and 7C show fluorescence images of the separation andtransfer of four proteins (protein G, OVA, BSA, and α-actinin) in asample. FIG. 8A shows a fluorescence image of the separation of the fourproteins into distinct bands in the separation gel. FIG. 8B shows afluorescence image of the transfer of the separated proteins from theseparation gel to the binding medium. FIG. 8C shows a fluorescence imageof the binding of the separated proteins to the binding medium.

In alkaline tricine-arginine (TA) buffer (pH 8.2), PA was hydrolyzed andhad a net negative charge. When biotinylated binding members of large Mrand low-pl point such as IgG (pl=5.5-8.0, Mr=150 kDa) andβ-galactosidase (pl=4.61, Mr=465 kDa) were copolymerized in the PA gelusing a streptavidin-acrylamide linker, the charge density wasincreased, which was evidenced by a stronger immobilization of thesample proteins. Electrotransfer was completed in about 42 s. Based onfluorescent intensity, significantly detectable amounts of separatedproteins were retained. Retention of separated proteins after theimmobilization was 75%, 77%, 65%, and 78% for protein G, OVA, BSA, andα-actinin, respectively. The near 1:1 correlation between separationpattern and immobilization was noted (e.g., separation resolutionbetween protein G and OVA was 1.41 before and after the blotting),indicating that protein transport was efficient.

Assay from sample loading to electrotransfer was completed in about 63s, which was approximately 10-fold less time than a conventional Westernblotting assay. After immobilization, horizontal electric field wascontinuously applied to wash off residual CTAB from the binding gel.Washing off residual CTAB may facilitate subsequent probing antibodyintroduction because the probing antibody may precipitate when exposedto CTAB. FIG. 9A shows a fluorescence image of the proteins (protein G,OVA, BSA, and α-actinin) immobilized in the binding medium. In thefollowing blocking step, open charge sites on the gel were blocked byelectrophoretically introducing 1% BSA solubilized in TA buffer toprevent non-specific antibody binding (FIG. 3E). Residual BSA was washedoff by applying a reverse electric field (FIG. 3F). After the blocking,an antibody probe conjugated with Alexa Fluor 568 (Invitrogen) wasintroduced and incubated for 10 min (FIG. 3G). The antibody probe waswashed off by applying a reverse field (FIG. 3H). The immunoaffinityprobe bound specifically to the immobilized target, as shown in FIG. 9B.800 nM of protein G was detected without the need for aprotein-renaturing step, which is typically required for conventionalWestern blotting using SDS-PAGE gel electrophoresis.

CONCLUSION

Microfluidic devices as disclosed herein allowed for deviceminiaturization and automation of the assay by using multi-channelvoltage/current control. In addition, the microfluidic assay performancewas also significantly better than typical SDS-PAGE Western blottingassays, with the microfluidic assay having sample consumption reduced byabout 100-1000 times (e.g., about 10 ng) and rapid completion times(about 2.5 hours vs. 1-2 days). The electrostatic interaction betweenthe sample analytes and the binding medium allowed for theelectrotransfer of all resolved protein to the binding medium. Incertain embodiments, copolymerization of matched-pair antibodies withthe binding medium to immobilize the separated protein targets was notrequired. Instead, immunoaffinity probes were introduced after theseparated were electrostatically immobilized in the binding medium. Insome cases, electrostatic immobilization of the separated proteinsfacilitated detection of the separated proteins through the use ofenzyme-linked secondary antibodies, which may be specifically bound tothe primary antibody bound to the analyte of interest. Secondarydetection antibodies may facilitate an increase in detectable signal,which may allow for an increase in the detection limit and label-freedetection. In addition, embodiments of the present disclosure that useCTAB-PAGE may facilitate immunoblotting without complex andtime-consuming protein renaturation.

Example 2

Experiments were performed using the same experimental protocol asExample 1 to determine the transfer efficiency and reproducibility ofthe assay. The binding member (e.g., capture protein) used in thefabrication of the binding medium of the microfluidic device was 150 kDaIgG (anti-C-reactive protein). A sample containing protein G (20 kDa),OVA (45 kDa), BSA (68 kDa), and α-actinin (95 kDa) was separated in theseparation medium and transferred to the binding medium. The separationstep was completed in 21 sec after loading the sample (FIG. 10A). FIG.10B shows a fluorescence image during the transfer of the separatedsample 35 s after sample loading. Transfer of the separated sample fromthe separation medium 1010 to the binding medium 1020 was completed in42 sec (e.g., 63 sec after sample loading) (FIG. 10C). Proteins wereretained by the binding medium 1020 even after washing the boundproteins with buffer, as shown in the fluorescence image in FIG. 10D,taken 139 after sample loading. Table 2 shows the separation resolution(SR) of the proteins in the sample during the transfer step (FIG. 10B)and after binding to the binding medium (FIG. 10C). As shown in Table 2,the proteins maintained their separation resolution during the transferand binding steps. Table 3 shows the capture efficiency (%) for theproteins in the sample after binding to the binding medium (FIG. 10C)and after the buffer wash (FIG. 10D). As shown in Table 3, the bindingmedium retained detectable amounts of each protein during the assay.

TABLE 2 Separation Resolution Assay Step Protein G-OVA OVA-BSABSA-α-actinin Transfer 1.41 0.90 1.32 Binding 1.41 0.97 1.76

TABLE 3 Capture Efficiency (%) Assay Step Protein G OVA BSA α-actininBinding 75 77 65 78 Buffer Wash 52 57 49 25

Example 3

Experiments were performed using the same experimental protocol asExample 1 to determine the transfer efficiency and reproducibility ofthe assay. The binding member (e.g., capture protein) used in thefabrication of the binding medium of the microfluidic device was 465 kDaβ-galactosidase (pL=4.61). A sample containing protein G (20 kDa), OVA(45 kDa), BSA (68 kDa), and α-actinin (95 kDa) was separated in theseparation medium and transferred to the binding medium. The separationstep was completed in 18 sec after loading the sample (FIG. 11A). FIG.11B shows a fluorescence image during the transfer of the separatedsample 21 s after sample loading. Transfer of the separated sample fromthe separation medium 1110 to the binding medium 1120 was completed in20 sec (e.g., 38 sec after sample loading) (FIG. 11C). Proteins wereretained by the binding medium 1120 even after washing the boundproteins with buffer, as shown in the fluorescence image in FIG. 11D,taken 53 s after sample loading. Table 4 shows the separation resolution(SR) of the proteins in the sample during the transfer step (FIG. 11B)and after binding to the binding medium (FIG. 11C). As shown in Table 4,the proteins maintained their separation resolution during the transferand binding steps. Table 5 shows the capture efficiency (%) for theproteins in the sample after binding to the binding medium (FIG. 11C)and after the buffer wash (FIG. 11D). As shown in Table 5, the bindingmedium retained detectable amounts of each protein during the assay.

TABLE 3 Separation Resolution Assay Step Protein G-OVA OVA-BSABSA-α-actinin Transfer 1.60 0.89 1.01 Binding 1.55 0.86 1.05

TABLE 4 Capture Efficiency (%) Assay Step Protein G OVA BSA α-actininBinding 71 79 62 — Buffer Wash 45 42 45 27

Example 4 Fabrication of Microfluidic Device

A glass microfluidic chip was designed using AUTOCAD software (Autodesk,San Rafael, Calif.), and micromachined (Caliper Life Sciences,Hopkinton, Mass.). The width of the injection channel was 25 μm, and thewidth of control channels was 10 μm. The size of the centralmicrofluidic chamber was 1.0 mm×1.5 mm (FIGS. 1A-1B). Eight access portswere drilled and the chip was thermally bonded to a blank chip in aprogrammable oven. The completed glass chip is shown in FIG. 1A. Theinterior of a glass chip was thoroughly cleaned before gelpolymerization using 0.1 M NaOH (10 min), DI water (10 min), andmethanol (5 min). The inner glass surface was functionalized forcovalent linkage to PA (polyacrylamide) gel using silane solution thatincluded a 2:2:3:3 ratio of 3-(trimethoxysilyl) propyl methacrylate,glacial acetic acid, DI water, and methanol (all from Sigma, St. Luis,Mo.). Gel precursor solution was introduced inside the glass chip bycapillary action (FIG. 6, Step 1). The electrostatic binding medium wasprepared by copolymerizing β-gal (β-galactosidase, tetramer, 465 kDa)with acrylamide using streptavidin-biotin linker. The precursorcomposition was 6-9% T w/v acrylamide/bisacrylamide (Sigma), 3.8 μM ofstreptavidin acrylamide (Invitrogen, Carlsbad, Calif.), 0-1.6 μM ofbiotinylated β-gal from E. coli (Sigma), and 0.1% w/v photoinitiatorVA-086 solution (Wako Chemical, Richmond, Va.) in 1×TA(tricine-arginine) buffer (Sigma). The PA precursor composition waspolymerized using an inverted microscope (IX-70, Olympus, Center Valley,Pa.), UV light source, and a photolithography mask (FIG. 6, Step 2).Un-polymerized precursor composition was evacuated and exchanged withsubsequent precursor using vacuum suction (FIG. 6, Step 3). After gelfabrication was completed, the chip was stored in 1×TA buffer until use.The microfluidic device was reusable; after the assay was completed, themicrofluidic chips were recycled by removing PA gels in heated 2:1perchloric acid and hydrogen peroxide bath.

Assay Steps

The method includes five assay steps: 1) sample loading; 2) CTAB-PAGEfor protein separation and sizing; 3) electrotransfer and electrostaticpan-capture of the separated proteins by the electrostatic bindingmedium (EBM); 4) blocking of non-specific binding sites in EBM; and 5)antibody-based probing of the immobilized protein bands. The assay maybe automated, and the voltage and current sequence of the assay stepsfor the microfluidic system is shown in Table 5, below.

TABLE 5 Voltage and Current Sequence Assay steps/ access port # 1 2 3 45 6 7 8 1) Sample loading −261 V −350 V −200 V    0 A    0 A    0 A    0A −267 V 2) Separation −200 V −320 V −320 V −270 V −270 V    0 A    0 A−400 V 3) Electrotransfer    0 A    0 A    0 A    0 A    0 A −200 V −100V    0 A 3) Residual CTAB wash    0 A    0 A    0 A    0 A    0 A −200 V−100 V    0 A 4) Blocking buffer injection    0 A    0 A    0 A    0 A   0 A −200 V −300 V    0 A 4) Residual blocking buffer    0 A    0 A   0 A    0 A    0 A −200 V −100 V    0 A wash 5) Antibody probeinjection    0 A    0 A    0 A    0 A    0 A −200 V −300 V    0 A 5)Residual antibody wash    0 A    0 A    0 A    0 A    0 A −200 V −100 V   0 A

A separation medium of 6% T PA gel was photopolymerized in the centralchamber (FIG. 2) and defined the separation axis. The relatively largerpore-size loading gel (3% T) allowed efficient protein loading, and thelarger-to-smaller pore-size at the interface between the loading medium(3% T) and the separation medium (6% T) minimized injection dispersionby “stacking” the sample proteins. The run buffer along the separationaxis was defined by electrophoretically injecting a stream of CTAB priorto protein injection (FIG. 4). CTAB detergent was included in the bufferof the loading medium and separation medium. CTAB was added to thebuffer in situ. CTAB of 0.1-0.5% was loaded to injection channels #1-3(see FIG. 2) and electrophoretically introduced into microchannels for10 min. Then, CTAB was injected to the separation medium as a narrowband (FIG. 4) for 10 min before the sample loading. This injectiondefined the separation axis with minimal protein band dispersion.

Protein G (PG, 20 kDa), OVA (45 kDa), BSA (68 kDa), (all fromInvitrogen), and phosphorylase b (97.2 kDa), α-actinin (αA, 95 kDa),β-gal* (β-galactosidase monomer, 116 kDa), (all from Sigma), and S100B(10.5 kDa) and lactoferrin (LF, 78 kDa), (all from Abcam, Cambridge,Mass.), were either conjugated as manufactured or conjugated before usewith Alexa Fluor 488 (Invitrogen) following manufacturer's instruction.Anti-rabbit polyclonal-PG (Abcam) was conjugated with Alexa Fluor 568(Invitrogen). The proteins and antibodies were all solubilized in 1×TAbuffer. Before the assay began, protein sample was diluted in samplebuffer (1×TA: 25 mM tricine+14 mM arginine+0.1% w/v CTAB) 15 min beforethe assay and then electrophoretically loaded to the loading medium.

In Step 1) of the assay, a protein sample that included β-gal* (*indicates β-gal monomer, not “β-gal”, the tetramer used for proteincapture in the EBM), BSA, OVA and protein G was injected along theseparation axis. In contrast to a typical SDS-PAGE system, positivelycharged CTAB-protein complexes migrated from the anode to the cathode.The discontinuous 3 to 6% T gel interface between the loading medium andthe separation medium reduced the injected protein sample plug width,defined as ±2σ of a Gaussian fit, by 57% (i.e., 493 to 213 μm).

During Step 2), CTAB-PAGE was used to separate the proteins and obtainprotein size information. Under an applied electric field (47 V/cm),intermediate to small Mr species were fully resolved (e.g., SR>1.5 forthe least resolved BSA-OVA peaks) after an elapsed separation time of 36s. Sizing was completed in a separation distance of 1496 μm (defined asthe fastest peak position when the least resolved peak pair is atSR=1.5). CTAB-PAGE analysis of a Mr standard ladder (20 to 118 kDa: 200nM each of protein G, OVA, BSA and α-actinin) yielded a log-linear Mr tomobility relation (y=−1.96×10³ _(X)+5.33, R²=0.997, n=5; see FIG. 7(bottom)).

The molecular mass of unknown species was also accurately predictedusing CTAB-PAGE. Using a log-linear Mr to mobility curve, Mr oflactoferrin (LF, 78 kDa) was estimated within 1.1% error. Five ladderproteins, S100B, OVA, BSA, phosphorylase b, and β-gal*, were used toyield a log-linear Mr to mobility relation. LF protein (78 kDa)co-migrated with the ladder, and Mr of the LF was calculated usingmobility data and the log-linear Mr to mobility calibration curve. Thepredicted Mr was 78.8 kDa (1.05% error).

Step 3) included transfer and binding, which included electrophoretictransfer of the separated proteins and pan-protein capture on theelectrostatic binding medium (EBM). The electrostatic capture wasnon-specific, meaning that all CTAB-protein complexes were immobilizedon the binding medium. Detectable labels (e.g., detection antibodies) infree solution were introduced to the binding medium afterelectrotransfer. No matched antibody pair was needed in the bindingmedium. To fabricate the EBM, β-galactosidase (β-gal) was copolymerizedin a 9% T PA gel as the anionic moiety. β-gal had a high negativesurface charge resulting from a low p/point (4.61) and large Mr (465kDa). The total number of negative surface charges on wild type β-galfrom E. coli was significant (−160, pH 8.2). Copolymerization of β-galat 1.6 μM with the PA binding medium yielded a stationary,charge-bearing region contiguous with the separation medium.

As shown in FIG. 12, resolved proteins were electrotransferred from theseparation axis to the EBM. Electrotransfer was completed in 31 s byapplying a transverse electric field. Electrostatic attraction to theEBM bound the CTAB-protein complexes to the binding medium and yieldedimmobilization of the proteins. In addition, protein bands migratinginto the EBM were enriched by being horizontally compressed (25%, 56%and 65% peak-width reduction for PG, OVA and BSA, respectively). Asdetermined by comparing the AUC (area under the curve) before transferto that on the EBM, material retention was 92%, 100%, 66% and 21% forPG, OVA, BSA and β-gal*, respectively at 31 s. PA gel hydrolyzes andbears net negative charges in alkaline media. Positively chargedCTAB-protein complexes bind to the negatively charged binding medium.Sample proteins were immobilized on the binding medium without loss ofseparation information, which facilitated subsequent identification ofthe separated proteins. Throughout Steps 4) and 5) afterelectrotransfer, sizing information was retained as shown in thecorresponding fluorescence intensity graphs in FIG. 12. Comparison ofseparation resolution (SR) on the separation axis to that on the EBMshows that the separation of the sample proteins was retained duringelectrotransfer and binding (see Table 6). Table 6 shows separationresolution between two neighboring protein peaks during microfluidicassay steps. Separation resolution was calculated using the equation:SR=(X₂−X₁)/(w₂+w₁), where X₁, X₂ are the center of two neighboring peaksand w₁, w₂ are their peak widths (2 times of variance of Gaussian fit).The peak width and center were estimated using nonlinear Gaussian curvefitting (OriginLab, Northampton, Mass.).

TABLE 6 Separation Resolution (SR) and Percent Variation (% V)Neighboring Protein Bands protein G - OVA OVA - BSA BSA - β-gal* AssayStep SR %V SR %V SR %V Separation 1.38 — 0.92 — 1.52 — Transfer 1.361.47 0.96 4.17 1.69 10.0 Binding 1.32 4.55 0.95 3.16 1.52 0 Washing 1.370.73 1.00 8 1.84 17.4 Blocking 1.31 5.34 1.02 9.80 1.50 1.33

Step 4) of the assay was an EBM blocking step that contacted the chargesites of the PA binding medium with BSA to minimize non-specific bindingof antibody probe in subsequent Step 5). Prior to blocking, residual andfree CTAB was electrophoretically washed from the EBM by applying alateral electric field for 30 min (E=45 V/cm). The washing stepminimized background signal that may be due to antibody association withCTAB. During the washing step, protein capture was 11%, 29%, 13% and 5%for PG, OVA, BSA and β-gal*, respectively. For the blocking step, 1% BSAw/v was electrophoretically contacted to the EBM using reverse polarity(e.g., BSA was negatively charged in non-detergent condition). After a10 min blocking incubation, a reverse field was applied for 15 min toremove unbound blocking BSA. Following the blocking step, immobilizedprotein signal was 6%, 19%, 8% and 5% for PG, OVA, BSA and β-gal*,respectively. After the final blocking-BSA wash, protein capture reacheda steady state. FIG. 13 shows a graph of fluorescence intensity(normalized peak height), representing material retention of proteinbands, over time (seconds). The fluorescence intensity decreasedexponentially as a function of time in the semi-log plot (the time axiswas shifted 0.1 s because of the logarithmic time scale). In FIG. 13, nofurther significant reduction was observed after the washing step toremove unbound blocking BSA.

In Step 5), antibody probing of the immobilized and blocked separatedproteins on the EBM was performed. Antibody probe conjugated with redfluorophore (Alexa Fluor 568) was electrophoretically introduced to theEBM, incubated for 10 min, and unbound antibody was electrophoreticallywashed away. FIG. 14 shows a multispectral fluorescence image of probingresults for 200 nM rabbit polyclonal antibody binding to an immobilizedprotein G (PG) target. Fluorescence signal from the probing antibody wasspecific for PG with a low background (SNR of 34, based on AUC) 11 minafter the antibody washing step. The signal-to-noise ratio (SNR) ofimmobilized proteins was still detectable after two hours of electricfield application: 6, 16, 8 and 5 for PG, OVA, BSA and β-gal*,respectively. Antibody probing of PG at two concentrations (600 nM vs.260 nM) resulted in corresponding probing antibody signals of 1278 vs.637, respectively. The direct correlation between concentration andsignal may facilitate development of a calibration curve, thus allowingabsolute protein quantitation. Antibody probing did not include aseparate protein renaturing step required for conventional SDS Westernblotting. Compared with conventional Western blotting, assay durationswere reduced about over 100× from protein loading to electrotransfer(about 3 min vs. 4-5 h), and 10 to 20× for the complete assay (e.g.,about 2 h vs. 1-2 days). Sample consumption was also reduced over 100×(e.g., about 10 ng vs. 1-40 μg).

Example 5

Additional experiments were performed to characterize protein capture onthe EBM. Two physicochemical EBM properties were studied: the chargedensity of capture sites on the EBM and the ionic strength of thetransfer buffer (see FIGS. 15A-15B). As a test sample, PG and BSA (+0.1%CTAB) were injected, separated, and immobilized in the EBM. Similar toelectrotransfer characterization done in Step 3) of Example 4 discussedabove, the capture efficiency was obtained based on material retentionafter 30 min of electric field application for the CTAB washing in Step4) of Example 4. To study the effect of varying the charge density,microfluidic devices having an EBM with an increasing immobilized β-galconcentration (0 μM, 0.2 μM, 0.8 μM, 1.6 μM) were fabricated (FIG. 15Aand FIG. 16). FIG. 16 shows fluorescence images of experiments studyingthe binding strength of proteins in different charge densities. ProteinG and BSA migrated as bands in the separation medium 1610. After atransverse electric field was applied, protein G and BSA bound withdifferent capture efficiencies to the binding medium 1620 in differentconcentrations of β-galactosidase (e.g., 0 μM, 0.2 μM, 0.8 μM, and 1.6μM). Binding in 1 mM Immobiline is also shown for comparison. Materialretention for both PG and BSA increased with increasing β-galconcentration up to 1.6 μM with PG reaching a maximum. Protein captureincreased with increasing charge on oppositely charged surfaces. Thelower capture efficiency of PG as compared to BSA may be due to PGhaving a lower Mr and surface charge. A weak interaction of BSA with theEBM was observed at 0 μM β-gal due to the PA gel having a negativecharge. At higher concentrations of β-gal (e.g., 3.2 μM) in the EBM,protein capture was reduced (data not shown).

Experiments were performed in which the transfer-buffer ionic strengthwas varied to study the impact of Debye length in the EBM pores onprotein capture (FIG. 15B). Three different ionic strength conditionswere tested: 115.4 mM, 16.4 mM and 4.5 mM for 7.57×, 1× and 0.13×TAbuffer+0.1% CTAB, respectively. As shown in FIG. 15B, protein capturedecreased with increasing buffer ionic strength and enhanced chargeshielding on the EBM. FIG. 17 shows fluorescence images of the bindingstrength of proteins in different ionic strength buffers (e.g.,different buffer concentrations). Binding for protein G and BSA is shownto the separation medium 1710 and the electrostatic binding medium 1720before and after binding (e.g., immobilization) to the binding medium indifferent buffer concentrations (e.g., 7.57×, 1× and 0.13×TA buffer+0.1%CTAB). As the Debye length decreased (e.g., increasing buffer ionicstrength), protein capture efficiencies decreased due to an enhancedcharge shielding effect.

Because CTAB association to proteins depended on buffer ionic strength,surface charge of the protein-CTAB complex also varied depending onionic strength. In order to decouple this effect of varying surfacecharge to the protein capture from varying ionic strength, surfacecharge densities of protein G and BSA were characterized. As relativeincrease or decrease of surface charge Q_(S)(C) at arbitrary bufferconcentration C is of interest, Q_(S)(C)/Q_(S)(C₀), relative chargedensity of CTAB-protein complexes relative to a standard concentrationC₀ (1×TA buffer) was obtained. Henry's equation for electrophoreticmobility, electrophoretic mobility data, and hydrodynamic parameters ofproteins were used in the calculation of Q_(S)(C)/Q_(S)(C₀). A proteinwas assumed to be a perfect sphere (r=hydrodynamic radius) and to haveevenly-distributed surface charges. 10×TA buffer was successivelydiluted to prepare 7.57×, 1×, and 0.13×TA buffer solutions, and 0.1% w/vCTAB was added to each buffer. Ionic strength was calculated as 115.4mM, 16.37 mM, and 4.52 mM respectively. Fluorescently labeled PG (605nM) and BSA (179 nM) was solubilized together in each buffer condition.Two proteins were separated in a double-T junction chip (Caliper LifeSciences) with uniform 6% T PA gel under a constant electric field.Electrophoretic mobility was obtained based on the speed of migration(i.e., time to travel a 2 mm distance in a straight microfluidicchannel). The ratio of mobilities at different ionic strengths wascalculated using a mathematical relationship between the free solutionmobility and % T value (e.g., 6% T). The % T term was cancelled and thenQ_(S)(C)/Q_(S)(C₀) was expressed as a function of free solutionmobility. Viscosity of the tricine-arginine buffer system was measuredusing a rotating rheometer (Physica MCR 301, Anton-Paar, Ashland, Va.).Stokes radius of BSA was obtained from literature. For the engineeredrecombinant PG (Invitrogen, 20 kDa), the radius was estimated fromStokes radii of proteins with similar Mr values. Finally, the relativenegative surface charge at 7.57× and 0.13×TA buffer, with respect to1×TA buffer, was calculated using Henry's description. Table 7 listsparameters used in the calculation, viscosity μ, experimentally obtainedmobility at 6% T gel n, ratio between hydrodynamic radius of proteins aand Debye length λ_(D), and surface charge ratio Q_(S)(C)/Q_(S)(C₀).

TABLE 7 Hydrodynamic property and surface charge ratios of CTAB-PG andCTAB-BSA complexes in three different buffer concentrations Protein GBSA Buffer μ η Q_(s)(C)/ η Q_(s)(C)/ Concentration (mPa · s)(10⁻⁵cm²s⁻¹V⁻¹) a/λ_(D) Q_(s)(C₀) (10⁻⁵cm²s⁻¹V⁻¹) a/λ_(D) Q_(s)(C₀) 7.57x 1.087 9.46 2.91 2.291 6.26 4.01 1.279 1 x 0.956 8.28 1.09 1 5.56 1.511 0.13 x 0.911 6.66 0.57 0.647 4.68 0.79 0.647

FIG. 15B indicates that protein capture decreased with increasing ionicstrength, which was the opposite effect observed due to surface chargeon protein capture. The decrease in protein capture with increasingionic strength indicated that electrostatic interaction was the dominantmechanism for protein capture on the EBM.

Example 6

Protein capture efficiency may be adjusted d by increasing the chargedensity and/or the decreasing buffer concentration. Additionalexperiments were performed to copolymerize a negatively charged bindingmember in the EBM. The negatively charged binding member used was anacrylamido buffer, an acidic Immobiline species (pK=3.8). Proteinimmobilization was observed using the Immobiline as the binding member.

Experiments that included Immobiline in the electrostatic binding medium(EBM) were performed as follows. An acidic Immobiline species (pK=3.8,GE healthcare, Pittsburgh, Pa.) was copolymerized with the EBM. The EBMwas fabricated with several different gel porosities (e.g., 6-10% T) andImmobiline concentrations (e.g., 0.1-10 mM), and capture efficiency wastested. Concentration polarization was observed at the EBM during theseparation step (E=87 V/cm) for the highest concentration Immobilineused (10 mM). With this EBM, the current from control channel #5 (seeFIG. 1B) decreased exponentially to less than 10% of the initial valueafter a few seconds. The decrease in current may be due to highstationary charge densities created at the interface between theseparation medium and the electrostatic binding medium. At 1 mMImmobiline, moderate concentration polarization was observed, andCTAB-protein complexes were sized and immobilized on the EBM. Thecapture efficiency at this Immobiline concentration was calculated to besimilar to that of the 0.2 μM β-gal copolymerized EBM, and was less thanthe capture efficiency using 1.6 μM of β-gal. The net charge density for0.2 μM β-gal in the EBM was estimated to be 0.032 mM. At 0.1 mMImmobiline concentration, the protein capture on the EBM was equivalentto the baseline response of the 0 μM β-gal copolymerized EBM. FIG. 18shows schematics of a comparison of charge interaction between aCTAB-protein complex and a) Immobilines, and b) β-gal copolymerized in apolyacrylamide gel pore. The pore geometry was idealized and Immobilinewas assumed to be evenly distributed. Based on dimensional analysis, oneto two molecules of β-gal were immobilized in each pore of a 6% T PA gelat 1.6 μM β-gal. β-gal molecules acted as a concentrated point charge(e.g., −160 per molecule). Charges originating from grafted Immobilinesmay be evenly distributed around each pore (FIG. 18). Thus,electrostatic attraction forces for Immobilines were in all directions,resulting in a vector force summation that may be less than theattraction towards point charges, such as β-gal.

Example 7

Immunoblotting assays were performed to study protein biomarkers ofdisease and dysfunction. A microfluidic assay was performed for theanalysis of lactoferrin (LF). LF is a biomarker for Sjögren's Syndrome,an autoimmune disease where immune cells attack exocrine glands.Microfluidic CTAB-PAGE analysis of LF in human tear fluid was performed.An increased CTAB concentration (0.5%) was used to prime the separationaxis. Higher CTAB concentrations may increase shielding of negativecharges on the PA separation gel, as LF was observed to non-specificallyassociate with the negatively charged separation medium. After anantibody screening, carrier-free anti-LF goat polyclonal antibody wasused to produce a detectable signal for LF. Diluted BSA (0.2%) was usedas the blocking buffer. FIG. 19( a) shows the results for a sample of LFpurified from human breast milk (400 nM) spiked into 1×TA buffer+0.2%CTAB along with a protein ladder (OVA and β-gal*). At 16 s elapsedseparation time (E=84 V/cm), CTAB-PAGE separated LF from the OVA andβ-gal* (SR>1.5). An unresolved peak (*) about 85 kDa migrated closelywith the LF peak. A control experiment showed the source of theunidentified peak as the β-gal* sample. Total separation time was 19 sand the total assay time, including antibody binding and detection, was115 min. After binding to the binding medium, the detectable labelantibody bound to LF (SNR=275, 5 min after starting the antibody washstep).

The assay was also performed on diluted tear fluid (48×) with 600 nM LFadded (FIG. 19( b)). CTAB in the sample buffer was increased to 0.7% tofacilitate detergent association due to the background of proteins intear fluid (about 80 species, total 10 mg/mL). Fluorescence signal of asample before separation was 31% of the signal for the case where notear fluid matrix was included in the sample. At 14 s of elapsedseparation time and 992 μm of separation length (E=60 V/cm), LF wasseparated from the co-migrating protein sample with the exception of theunidentified peak (*). Binding with antibody specific to LF wascoincident with the LF band observed during CTAB-PAGE. SNR of theimmunoblot to LF was 43 (5 min after starting the antibody wash step).Total separation time was 21 s and the total assay time, includingantibody binding and detection, was 115 min.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of the present disclosure that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A microfluidic device for detecting an analyte in a fluid sample,wherein the microfluidic device comprises: a separation medium having aseparation flow path with a first directional axis; and a pan-capturebinding medium in fluid communication with the separation medium andhaving a flow path with a second directional axis, wherein themicrofluidic device is configured to subject a sample to two or moredirectionally distinct flow fields.
 2. The microfluidic device accordingto claim 1, wherein the binding medium is configured to non-specificallybind to analytes in the sample through electrostatic interactions. 3.The microfluidic device according to claim 1, wherein the binding mediumis configured to have a negative charge.
 4. The microfluidic deviceaccording to claim 3, wherein the binding medium comprises a negativelycharged gel.
 5. The microfluidic device according to claim 3, whereinthe binding medium comprises a negatively charged pan-capture bindingmember stably associated with a support.
 6. The method according toclaim 1, wherein the fluid sample comprises a detergent configured toprovide analytes in the sample with a positive charge.
 7. The methodaccording to claim 6, wherein the detergent comprisescetyltrimethylammonium bromide.
 8. The microfluidic device according toclaim 1, wherein the analyte comprises a fluorescent label.
 9. Themicrofluidic device according to claim 1, wherein the two or moredirectionally distinct flow fields comprise two or more directionallydistinct electric fields.
 10. The microfluidic device according to claim1, wherein the second directional axis orthogonal to the firstdirectional axis.
 11. The microfluidic device according to claim 1,wherein the microfluidic device comprises a chamber containing theseparation medium and the binding medium.
 12. A method of detecting ananalyte in a fluid sample, the method comprising: (a) introducing thefluid sample comprising the analyte into a microfluidic deviceconfigured to subject the sample to two or more directionally distinctflow fields, wherein the microfluidic device comprises: (i) a separationmedium having a separation flow path with a first directional axis; and(ii) a pan-capture binding medium in fluid communication with theseparation medium and having a flow path with a second directional axis;(b) directing the sample through the separation medium to produce aseparated sample; and (c) detecting the analyte in the separated sample.13. The method according to claim 12, further comprising transferringthe separated sample to the binding medium.
 14. The method according toclaim 13, further comprising contacting the analyte with a label thatspecifically binds to the analyte to produce a labeled analyte.
 15. Themethod according to claim 14, further comprising detecting the labeledanalyte.
 16. The method according to claim 12, wherein the two or moredirectionally distinct flow fields comprise two or more directionallydistinct electric fields.
 17. The method according to claim 12, whereinthe method is a diagnostic method.
 18. The method according to claim 12,wherein the method is a validation method.
 19. A system for detecting ananalyte in a fluid sample, the system comprising: (a) a microfluidicdevice configured to subject a sample to two or more directionallydistinct flow fields, wherein the microfluidic device comprises: (i) aseparation medium having a separation flow path with a first directionalaxis; and (ii) a pan-capture binding medium in fluid communication withthe separation medium and having a flow path with a second directionalaxis; and (b) a detector. 20-25. (canceled)